Boronate ester compounds and pharmaceutical compositions thereof

ABSTRACT

The present invention provides novel compounds useful as proteasome inhibitors. The invention also provides pharmaceutical compositions comprising the compounds of the invention and methods of using the compositions in the treatment of various diseases.

This application is a divisional of U.S. Utility patent application Ser.No. 14/449,328 filed Aug. 1, 2014, which is a divisional of U.S. Utilitypatent application Ser. No. 13/667,164, filed Nov. 2, 2012, which is acontinuation of U.S. utility patent application Ser. No. 12/485,344,filed Jun. 16, 2009, which claims priority from U.S. Provisional PatentApplication Ser. No. 61/132,244, filed Jun. 17, 2008, and U.S.Provisional Patent Application Ser. No. 61/211,499, filed Mar. 31, 2009;both which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to boronate ester compounds useful asproteasome inhibitors. The invention also provides pharmaceuticalcompositions comprising the compounds of the invention, and methods ofusing the compositions in the treatment of various diseases.

BACKGROUND OF THE INVENTION

Boronic acid and boronate ester compounds display a variety ofpharmaceutically useful biological activities. Shenvi et al., U.S. Pat.No. 4,499,082 (1985), discloses that peptide boronic acids areinhibitors of certain proteolytic enzymes. Kettner and Shenvi, U.S. Pat.No. 5,187,157 (1993), U.S. Pat. No. 5,242,904 (1993), and U.S. Pat. No.5,250,720 (1993), describe a class of peptide boronic acids that inhibittrypsin-like proteases. Kleeman et al., U.S. Pat. No. 5,169,841 (1992),discloses N-terminally modified peptide boronic acids that inhibit theaction of renin. Kinder et al., U.S. Pat. No. 5,106,948 (1992),discloses that certain boronic acid compounds inhibit the growth ofcancer cells. Magde et al., WO 04/022070 discloses peptide boronic acidcompounds that inhibit thrombin. Boucher, U.S. Patent Application Pub.No. 2006/0084592 discloses various basic addition salts of peptideboronic acid compounds. Bachovchin et al., WO 07/005991, disclosespeptide boronic acid compounds that inhibit fibroblast activatingprotein.

Boronic acid and ester compounds hold particular promise as inhibitorsof the proteasome, a multicatalytic protease responsible for themajority of intracellular protein turnover. Adams et al., U.S. Pat. No.5,780,454 (1998), describes peptide boronic ester and acid compoundsuseful as proteasome inhibitors. The reference also describes the use ofboronic ester and acid compounds to reduce the rate of muscle proteindegradation, to reduce the activity of NF-κB in a cell, to reduce therate of degradation of p53 protein in a cell, to inhibit cyclindegradation in a cell, to inhibit the growth of a cancer cell, and toinhibit NF-κB dependent cell adhesion. Furet et al., WO 02/096933,Chatterjee et at, WO 05/016859, and Bernadini et al, WO 05/021558 and WO06/08660, disclose additional boronic ester and acid compounds that arereported to have proteasome inhibitory activity.

Ciechanover, Cell, 79:13-21 (1994), discloses that the proteasome is theproteolytic component of the ubiquitin-proteasome pathway, in whichproteins are targeted for degradation by conjugation to multiplemolecules of ubiquitin. Ciechanover also discloses that theubiquitin-proteasome pathway plays a key role in a variety of importantphysiological processes. Rivett et al., Biochem. J. 291:1 (1993)discloses that the proteasome displays tryptic-, chymotryptic-, andpeptidylglutamyl-peptidase activities. Constituting the catalytic coreof the 26S proteasome is the 20S proteasome. McCormack et al.,Biochemistry 37:7792 (1998), teaches that a variety of peptidesubstrates, including Suc-Leu-Leu-Val-Tyr-AMC, Z-Leu-Leu-Arg-AMC, andZ-Leu-Leu-Glu-2NA, wherein Suc is N-succinyl, AMC is7-amino-4-methylcoumarin, and 2NA is 2-naphthylamine, are cleaved by the20S proteasome.

Proteasome inhibition represents an important new strategy in cancertreatment. King et al., Science 274:1652-1659 (1996), describes anessential role for the ubiquitin-proteasome pathway in regulating cellcycle, neoplastic growth and metastasis. The authors teach that a numberof key regulatory proteins, including, cyclins, and the cyclin-dependentkinases p21 and p27^(KIP1), are temporally degraded during the cellcycle by the ubiquitin-proteasome pathway. The ordered degradation ofthese proteins is required for the cell to progress through the cellcycle and to undergo mitosis.

Furthermore, the ubiquitin-proteasome pathway is required fortranscriptional regulation. Palombella et al., Cell, 78:773 (1994),teaches that the activation of the transcription factor NF-κB isregulated by proteasome-mediated degradation of the inhibitor proteinIκB. In turn, NF-κB plays a central role in the regulation of genesinvolved in the immune and inflammatory responses. Read et al., Immunity2:493-506 (1995), teaches that the ubiquitin-proteasome pathway isrequired for expression of cell adhesion molecules, such as E-selectin,ICAM-1, and VCAM-1. Zetter, Seminars in Cancer Biology 4:219-229 (1993),teaches that cell adhesion molecules are involved in tumor metastasisand angiogenesis in vivo, by directing the adhesion and extravasation oftumor cells to and from the vasculature to distant tissue sites withinthe body. Moreover, Beg and Baltimore, Science 274:782 (1996), teachesthat NF-κB is an anti-apoptotic controlling factor, and inhibition ofNF-κB activation makes cells more sensitive to environmental stress andcytotoxic agents.

The proteasome inhibitor VELCADE® (bortezomib;N-2-pyrazinecarbonyl-L-phenylalanine-L-leucineboronic acid) is the firstproteasome inhibitor to achieve regulatory approval. Mitsiades et al.,Current Drug Targets, 7:1341 (2006), reviews the clinical studiesleading to the approval of bortezomib for the treatment of multiplemyeloma patients who have received at least one prior therapy. Fisher etal., J. Clin. Oncol., 30:4867 (2006), describes an internationalmulti-center Phase II study confirming the activity of bortezomib inpatients with relapsed or refractory mantle cell lymphoma. Ishii et al.,Anti-Cancer Agents in Medicinal Chemistry, 7:359 (2007), and Roccaro etal., Curr. Pharm. Biotech., 7:1341 (2006), discuss a number of molecularmechanisms that may contribute to the antitumor activities ofbortezomib.

Structural analysis reported by Voges et al., Anna. Rev. Biochem.,68:1015 (1999) reveals that the 20S proteasome comprises 28 subunits,with the catalytic subunits β1, β2, and β5 being responsible forpeptidylglutamyl, tryptic, and chymotryptic peptidase activity,respectively. Rivett et al., Curr. Protein Pept. Sci., 5:153 (2004)discloses that when the proteasome is exposed to certain cytokines,including IFN-γ and TNF-α, the β1, β2, and β5 subunits are replaced withalternate catalytic subunits, β1i, β2i, and β5i, to form a variant formof the proteasome known as the immunoproteasome.

Orlowski, Hematology (Am. Soc. Hematol. Educ. Program) 220 (2005),discloses that the immunoproteasome also is expressed constitutively insome cells derived from hematopoietic precursors. The author suggeststhat inhibitors specific for the immunmoproteasome may allow fortargeted therapy against cancers arising from hematologic origins,thereby potentially sparing normal tissues, such as gastrointestinal andneurological tissues, from side effects.

Unfortunately, boronic acid compounds are relatively difficult to obtainin analytically pure form. For example, Snyder et al., J. Am. Chem. Soc.80: 3611 (1958), teaches that arylboronic acid compounds readily formcyclic trimeric anhydrides under dehydrating conditions. Also,alkylboronic acids and their boroxines are often air-sensitive. Korceket al., J. Chem. Soc., Perkin Trans. 2 242 (1972), teaches thatbutylboronic acid is readily oxidized by air to generate 1-butanol andboric acid. These difficulties limit the pharmaceutical utility ofboronic acid compounds, complicating the characterization ofpharmaceutical agents comprising boronic acid compounds and limitingtheir shelf-life.

Plamondon et al., WO 02/059131 discloses stable, pharmaceuticallyacceptable compositions prepared from boronic acid compounds and sugars.There remains a need for additional stable formulations of boronic acidcompounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a powder X-ray diffractogram of4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid (I-1) Form 1.

FIG. 2 is a differential scanning calorimetry (DSC)/thermal gravimetricanalysis (TGA) profile for4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid (I-1) Form 1.

FIG. 3 is a powder X-ray diffractogram of4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid (I-1) Form 2.

FIG. 4 is a differential scanning calorimetry (DSC)/thermal gravimetricanalysis (TGA) profile for4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid (I-1) Form 2.

FIG. 5 is a powder X-ray diffractogram of2,5-dichloro-N-[2-({(1R)-3-methyl-1-[(4S)-4-methyl-5-oxo-1,3,2-dioxaborolan-2-yl]butyl}amino)-2-oxoethyl]benzamide(I-7).

FIG. 6 is a powder X-ray diffractogram of2,5-dichloro-N-(2-{[(1R)-3-methyl-1-(4-oxo-4H-1,3,2-benzodioxaborinin-2-yl)butyl]amino}-2-oxoethyl)benzamide(I-13).

FIG. 7 is a powder X-ray diffractogram of4-(R,S)-carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid (I-1) Form 2.

FIG. 8 is a differential scanning calorimetry (DSC) profile of4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid (I-1) Form 2.

DESCRIPTION OF THE INVENTION

The present invention provides novel boronate ester compounds and stablepharmaceutically acceptable compositions comprising them. Thesecompounds and compositions are useful for inhibiting proteasome activityin vitro and in vivo, and are especially useful for the treatment ofvarious cell proliferative diseases.

In one aspect, the invention provides compounds of the general formula(I):

or a pharmaceutically acceptable salt or thereof wherein:

-   -   A is 0, 1, or 2;    -   P is hydrogen or an amino-group-blocking moiety;    -   R^(a) is hydrogen, C₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic,        —(CH₂)_(m)—CH₂—R^(B), —(CH₂)_(m)—CH₂—NHC(═NR⁴)NH-γ,        —(CH₂)_(m)—CH—CON(R⁴)₂, —(CH₂)_(m)—CH₂—N(R⁴)CON(R⁴)₂,        —(CH₂)_(m)—CH(R⁶)N(R⁴)₂, —(CH₂)_(m)—CH(R^(5a))—OR^(5b), or        —(CH₂)_(m)—CH(R⁵)—SR⁵;    -   R^(a1) is hydrogen, C₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic,        —(CH₂)_(m)—CH₂—R^(B), —(CH₂)_(m)—CH₂—NHC(═NR⁴)NH—Y,        —(CH₂)_(m)—CH₂—CON(R⁴)₂, —(CH₂)_(m)—CH₂—N(R⁴)CON(R⁴)₂,        —(CH₂)_(m)—CH(R⁶)N(R⁴)₂, —(CH₂)_(m)—CH(R^(5a))—OR^(5b), or        —(CH₂)_(m)—CH(R⁵)—SR⁵;    -   each R^(a2) independently is hydrogen, C₁₋₆ aliphatic, C₁₋₆        fluoroaliphatic, —(CH₂)_(m)—CH₂—R^(B),        —(CH₂)_(m)—CH₂—NHC(═NR⁴)NH—Y, —(CH₂)_(m)—CH₂—CON(R⁴)₂,        —(CH₂)_(m)—CH₂—N(R⁴)CON(R⁴)₂, —(CH₂)_(m)—CH(R⁶)N(R⁴)₂,        —(CH₂)_(m)—CH(R^(5a))—OR^(5b), or —(CH₂)_(m)—CH(R⁵)—SR⁵;    -   each R^(B) independently is a substituted or unsubstituted mono-        or bicyclic ring system;    -   each R⁴ independently is hydrogen or a substituted or        unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl        group; or two R⁴ on the same nitrogen atom, taken together with        the nitrogen atom, form a substituted or unsubstituted 4- to        8-membered heterocyclyl ring having, in addition to the nitrogen        atom, 0-2 ring heteroatoms independently selected from the group        consisting of N, O, and S;    -   each R⁵ independently is hydrogen or a substituted or        unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl        group;    -   each R^(5a) independently is hydrogen or a substituted or        unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl        group;    -   each R^(5b) independently is hydrogen or a substituted or        unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl        group;    -   each R⁶ independently is a substituted or unsubstituted        aliphatic, aryl, or heteroaryl group;    -   Y is hydrogen, —CN, or —NO₂;    -   m is 0, 1, or 2; and    -   Z¹ and Z² together form a moiety derived from an alpha-hydroxy        carboxylic acid, wherein the atom attached to boron in each case        is an oxygen atom; or Z¹ and Z² together form a moiety derived        from a beta-hydroxy carboxylic acid, wherein the atom attached        to boron in each case is an oxygen atom.

In another aspect, the present invention provides pharmaceuticalcompositions comprising the compound of formula (I), or a crystallineform thereof, and additional excipients described herein, suitable forthe production of an oral pharmaceutical dosage form.

In another aspect, the invention provides a pharmaceutical compositioncomprising the compound of formula (I), or a crystalline form thereof,and additional excipients described herein, suitable for the productionof a lyophilized powder pharmaceutical dosage form.

In another aspect, the invention provides a pharmaceutical compositioncomprising the compound of formula (I), or a crystalline form thereof,and additional excipients described herein, suitable for the productionof a liquid pharmaceutical dosage form.

In another aspect, the invention provides a pharmaceutical composition,comprising the compound of formula (I), or a crystalline form thereof, afiller, and optionally a lubricant.

In another aspect, the invention provides a pharmaceutical composition,comprising the compound of formula (I), or a crystalline form thereof afiller, optionally a lubricant, optionally a flow-aid, and optionally abuffer.

In another aspect, the invention provides a pharmaceutical composition,comprising the compound of formula (I), or a crystalline form thereof, abulking agent, and a buffer.

In another aspect, the invention provides processes for the productionof the pharmaceutical compositions of the invention.

In another aspect, the invention provides methods for the use of thepharmaceutical compositions of the invention, for treating a patienthaving, or at risk of developing or experiencing a recurrence of, aproteasome-mediated disorder.

In another aspect, the invention provides methods for the use of thepharmaceutical compositions of the invention for the treatment ofcancer.

Definitions

Unless otherwise explicitly stated, the term “proteasome” is intended torefer to both constitutive proteasome and immunoproteasome.

The term “aliphatic” or “aliphatic group”, as used herein, means asubstituted or unsubstituted straight-chain, branched, or cyclic C₁₋₁₂hydrocarbon, which is completely saturated or which contains one or moreunits of unsaturation, but which is not aromatic. For example, suitablealiphatic groups include substituted or unsubstituted linear, branchedor cyclic alkyl, alkenyl, or alkynyl groups and hybrids thereof, such as(cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. Invarious embodiments, the aliphatic group has 1 to 12, 1 to 8, 1 to 6, 1to 4, or 1 to 3 carbons.

The terms “alkyl”, “alkenyl”, and “alkynyl”, used alone or as part of alarger moiety, refer to a straight or branched chain aliphatic grouphaving from 1 to 12 carbon atoms. For purposes of the present invention,the term “alkyl” will be used when the carbon atom attaching thealiphatic group to the rest of the molecule is a saturated carbon atom.However, an alkyl group may include unsaturation at other carbon atoms.Thus, alkyl groups include, without limitation, methyl, ethyl, propyl,allyl, propargyl, butyl, pentyl, and hexyl.

For purposes of the present invention, the term “alkenyl” will be usedwhen the carbon atom attaching the aliphatic group to the rest of themolecule forms part of a carbon-carbon double bond. Alkenyl groupsinclude, without limitation, vinyl, 1-propenyl, 1-butenyl, 1-pentenyl,and 1-hexenyl.

For purposes of the present invention, the term “alkynyl” will be usedwhen the carbon atom attaching the aliphatic group to the rest of themolecule forms part of a carbon-carbon triple bond. Alkynyl groupsinclude, without limitation, ethynyl, 1-propynyl, 1-butynyl, 1-pentynyl,and 1-hexynyl.

The term “cycloaliphatic”, used alone or as part of a larger moiety,refers to a saturated or partially unsaturated cyclic aliphatic ringsystem having from 3 to about 14 members, wherein the aliphatic ringsystem is optionally substituted. In some embodiments, thecycloaliphatic is a monocyclic hydrocarbon having 3-8 or 3-6 ring carbonatoms. Nonlimiting examples include cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl,cycloheptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl. In someembodiments, the cycloaliphatic is a bridged or fused bicyclichydrocarbon having 6-12, 6-10, or 6-8 ring carbon atoms, wherein anyindividual ring in the bicyclic ring system has 3-8 members.

In some embodiments, two adjacent substituents on the cycloaliphaticring, taken together with the intervening ring atoms, form an optionallysubstituted fused 5- to 6-membered aromatic or 3- to 8-memberednon-aromatic ring having 0-3 ring heteroatoms selected from the groupconsisting of O, N, and S. Thus, the term “cycloaliphatic” includesaliphatic rings that are fused to one or more aryl, heteroaryl, orheterocyclyl rings. Nonlimiting examples include indanyl,5,6,7,8-tetrahydroquinoxalinyl, decahydronaphthyl, ortetrahydronaphthyl, where the radical or point of attachment is on thealiphatic ring.

The terms “aryl” and “ar-”, used alone or as part of a larger moiety,e.g., “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refer to a C₆ to C₁₄aromatic hydrocarbon, comprising one to three rings, each of which isoptionally substituted. Preferably, the aryl group is a C₆₋₁₀ arylgroup. Aryl groups include, without limitation, phenyl, naphthyl, andanthracenyl. In some embodiments, two adjacent substituents on the arylring, taken together with the intervening ring atoms, form an optionallysubstituted fused 5- to 6-membered aromatic or 4- to 8-memberednon-aromatic ring having 0-3 ring heteroatoms selected from the groupconsisting of O, N, and S. Thus, the term “aryl”, as used herein,includes groups in which an aryl ring is fused to one or moreheteroaryl, cycloaliphatic, or heterocyclyl rings, where the radical orpoint of attachment is on the aromatic ring. Nonlimiting examples ofsuch fused ring systems include indolyl, isoindolyl, benzothienyl,benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl,quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl,phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, fluorenyl,indanyl, phenanthridinyl, tetrahydronaphthyl, indolinyl, phenoxazinyl,benzodioxanyl, and benzodioxolyl. An aryl group may be mono-, bi-, tri-,or polycyclic, preferably mono-, bi-, or tricyclic, more preferablymono- or bicyclic. The term “aryl” may be used interchangeably with theterms “aryl group”, “aryl moiety”, and “aryl ring”.

An “aralkyl” or “arylalkyl” group comprises an aryl group covalentlyattached to an alkyl group, either of which independently is optionallysubstituted. Preferably, the aralkyl group is C₆₋₁₀ aryl(C₁₋₆)-alkyl,C₆₋₁₀ aryl(C₁₋₄)alkyl, or C₆₋₁₀ aryl(C₁₋₃)alkyl, including, withoutlimitation, benzyl, phenethyl, and naphthylmethyl.

The terms “heteroaryl” and “heteroar-”, used alone or as part of alarger moiety, e.g., heteroaralkyl, or “heteroaralkoxy”, refer to groupshaving 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having6, 10, or 14 π electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to four heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Thus, when used in reference to a ring atom of a heteroaryl,the term “nitrogen” includes an oxidized nitrogen (as in pyridineN-oxide). Certain nitrogen atoms of 5-membered heteroaryl groups alsoare substitutable, as further defined below. Heteroaryl groups include,without limitation, radicals derived from thiophene, furan, pyrrole,imidazole, pyrazole, triazole, tetrazole, oxazole, isoxazole,oxadiazole, thiazole, isothiazole, thiadiazole, pyridine, pyridazine,pyrimidine, pyrazine, indolizine, naphthyridine, pteridine,pyrrolopyridine, imidazopyridine, oxazolopyridine, thiazolopyridine,triazolopyridine, pyrrolopyrimidine, purine, and triazolopyrimidine. Asused herein, the phrase “radical derived from” means a monovalentradical produced by removal of a hydrogen radical from the parentheteroaromatic ring system. The radical (i.e., the point of attachmentof the heteroaryl to the rest of the molecule) may be created at anysubstitutable position on any ring of the parent heteroaryl ring system.

In some embodiments, two adjacent substituents on the heteroaryl, takentogether with the intervening ring atoms, form an optionally substitutedfused 5- to 6-membered aromatic or 4- to 8-membered non-aromatic ringhaving 0-3 ring heteroatoms selected from the group consisting of O, N,and S. Thus, the terms “heteroaryl” and “heteroar-”, as used herein,also include groups in which a heteroaromatic ring is fused to one ormore aryl, cycloaliphatic, or heterocyclyl rings, where the radical orpoint of attachment is on the heteroaromatic ring. Nonlimiting examplesinclude indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl,indazolyl, benzimidazolyl, benzthiazolyl, benzoxazolyl, quinolyl,isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl,phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, andpyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be mono-,bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, morepreferably mono- or bicyclic. The term “heteroaryl” may be usedinterchangeably with the terms “heteroaryl ring”, or “heteroaryl group”,any of which terms include rings that are optionally substituted. Theterm “heteroaralkyl” refers to an alkyl group substituted by aheteroaryl, wherein the alkyl and heteroaryl portions independently areoptionally substituted.

As used herein, the terms “aromatic ring” and “aromatic ring system”refer to an optionally substituted mono-, bi-, or tricyclic group having0-6, preferably 0-4 ring heteroatoms, and having 6, 10, or 14 πelectrons shared in a cyclic array. Thus, the terms “aromatic ring” and“aromatic ring system” encompass both aryl and heteroaryl groups.

As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclicradical”, and “heterocyclic ring” are used interchangeably and refer toa stable 3- to 7-membered monocyclic, or to a fused 7- to 10-membered orbridged 6- to 10-membered bicyclic heterocyclic moiety that is eithersaturated or partially unsaturated, and having, in addition to carbonatoms, one or more, preferably one to four, heteroatoms, as definedabove. When used in reference to a ring atom of a heterocycle, the term“nitrogen” includes a substituted nitrogen. As an example, in aheterocyclyl ring having 1-3 heteroatoms selected from the groupconsisting of oxygen, sulfur or nitrogen, the nitrogen may be N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or ⁺NR (as inN-substituted pyrrolidinyl). A heterocyclic ring can be attached to itspendant group at any heteroatom or carbon atom that results in a stablestructure, and any of the ring atoms can be optionally substituted.Examples of such saturated or partially unsaturated heterocyclicradicals include, without limitation, tetrahydrofuranyl,tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl,thiazepinyl, morpholinyl, and quinuclidinyl.

In some embodiments, two adjacent substituents on a heterocyclic ring,taken together with the intervening ring atoms, form an optionallysubstituted fused 5- to 6-membered aromatic or 3- to 8-memberednon-aromatic ring having 0-3 ring heteroatoms selected from the groupconsisting of O, N, and S. Thus, the terms “heterocycle”,“heterocyclyl”, “heterocyclyl ring”, “heterocyclic group”, “heterocyclicmoiety”, and “heterocyclic radical”, are used interchangeably herein,and include groups in which a heterocyclyl ring is fused to one or morearyl, heteroaryl, or cycloaliphatic rings, such as indolinyl,3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, wherethe radical or point of attachment is on the heterocyclyl ring. Aheterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferablymono-, bi-, or tricyclic, more preferably mono- or bicyclic. The term“heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond between ring atoms. Theterm “partially unsaturated” is intended to encompass rings havingmultiple sites of unsaturation, but is not intended to include aryl orheteroaryl moieties, as herein defined.

The terms “haloaliphatic”, “haloalkyl”, “haloalkenyl” and “haloalkoxy”refer to an aliphatic, alkyl, alkenyl or alkoxy group, as the case maybe, which is substituted with one or more halogen atoms. As used herein,the term “halogen” or “halo” means F, Cl, Br, or I. The term“fluoroaliphatic” refers to a haloaliphatic wherein the halogen isfluoro, including perfluorinated aliphatic groups. Examples offluoroaliphatic groups include, without limitation, fluoromethyl,difluoromethyl, trifluoromethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl,1,1,2-trifluoroethyl, 1,2,2-trifluoroethyl, and pentafluoroethyl.

The term “linker group” or “linker” means an organic moiety thatconnects two parts of a compound. Linkers typically comprise an atomsuch as oxygen or sulfur, a unit such as —NH—, —CH₂—, —C(O)—, —C(O)NH—,or a chain of atoms, such as an alkylene chain. The molecular mass of alinker is typically in the range of about 14 to 200, preferably in therange of 14 to 96 with a length of up to about six atoms. In someembodiments, the linker is a C₁₋₆ alkylene chain.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(y)—, wherein y is apositive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylenegroup in which one or more methylene hydrogen atoms is replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group. An alkylene chain also may be substitutedat one or more positions with an aliphatic group or a substitutedaliphatic group.

An alkylene chain also can be optionally interrupted by a functionalgroup. An alkylene chain is “interrupted” by a functional group when aninternal methylene unit is replaced with the functional group. Examplesof suitable “interrupting functional groups” include —C(R*)═C(R*)—,—C≡C—, —O—, —S—, —S(O)—, —S(O)₂—, —S(O)₂N(R⁺)—, —N(R*)—, —N(R⁺)CO—,—N(R⁺)C(O)N(R⁺)—, —N(R⁺)C(═NR⁺)—N(R⁺)—, —N(R⁺)—C(═NR⁺)—, —N(R⁺)CO₂—,—N(R⁺)SO₂—, —N(R⁺)SO₂N(R⁺)—, —OC(O), —OC(O)O—, —OC(O)N(R⁺)—, —C(O)—,—CO₂—, —C(O)N(R⁺)—, —C(O)—C(O)—, —C(═NR⁺)—N(R⁺)—, —C(NR⁺)═N—,—C(═NR⁺)—O—, —C(OR*)═N—, —C(R^(◯))—═N—O—, or —N(R⁺)—N(R⁺)—. Each R⁺independently is hydrogen or an optionally substituted aliphatic, aryl,heteroaryl, or heterocyclyl group, or two R⁺ on the same nitrogen atom,taken together with the nitrogen atom, form a 5-8 membered aromatic ornon-aromatic ring having, in addition to the nitrogen atom, 0-2 ringheteroatoms selected from the group consisting of N, O, and S. Each R*independently is hydrogen or an optionally substituted aliphatic, aryl,heteroaryl, or heterocyclyl group. Each R^(◯) independently is anoptionally substituted aliphatic, aryl, or heteroaryl group.

Examples of C₃₋₆ alkylene chains that have been “interrupted” with —O—include —CH₂OCH₂—, —CH₂O(CH₂)₂—, —CH₂O(CH₂)₃—, —CH₂O(CH₂)₄—,—(CH₂)₂OCH₂—, —(CH₂)₂O(CH₂)₂—, —(CH₂)₂O(CH₂)₃—, —(CH₂)₃O(CH₂)—,—(CH₂)₃O(CH₂)₂—, and —(CH₂)₄O(CH₂)—. Other examples of alkylene chainsthat are “interrupted” with functional groups include —CH₂Z*CH₂—,—CH₂Z*(CH₂)₂—, —CH₂Z*(CH₂)₃—, —CH₂Z*(CH₂)₄—, —(CH₂)₂Z*CH₂—,—(CH₂)₂Z*(CH₂)₂—, —(CH₂)₂Z*(CH₂)₃—, —(CH₂)₃Z*(CH₂)—, —(CH₂)₃Z*(CH₂)—₂—,and —(CH₂)₄Z*(CH₂)—, wherein Z* is one of the “interrupting” functionalgroups listed above.

One of ordinary skill in the art will recognize that when an alkylenechain having an interruption is attached to a functional group, certaincombinations would not be sufficiently stable for pharmaceutical use.Only stable or chemically feasible compounds are within the scope of thepresent invention. A stable or chemically feasible compound is one whichmaintains its integrity long enough to be useful for therapeutic orprophylactic administration to a patient. Preferably, the chemicalstructure is not substantially altered when kept at a temperature below−70° C., below −50° C., below −20° C., below 0° C., or below 20° C., inthe absence of moisture or other chemically reactive conditions for atleast a week.

The term “substituted”, as used herein, means that a hydrogen radical ofthe designated moiety is replaced with the radical of a specifiedsubstituent, provided that the substitution results in a stable orchemically feasible compound. The term “substitutable”, when used inreference to a designated atom, means that attached to the atom is ahydrogen radical, which can be replaced with the radical of a suitablesubstituent.

The phrase “one or more substituents”, as used herein, refers to anumber of substituents that equals from one to the maximum number ofsubstituents possible based on the number of available bonding sites,provided that the above conditions of stability and chemical feasibilityare met. Unless otherwise indicated, an optionally substituted group mayhave a substituent at each substitutable position of the group, and thesubstituents may be either the same or different.

As used herein, the terms “independently” or “independently selected”means that the same or different values may be selected for multipleinstances of a given variable in a single compound.

An aryl (including the aryl moiety in aralkyl, aralkoxy, aryloxyalkyland the like) or heteroaryl (including the heteroaryl moiety inheteroaralkyl and heteroaralkoxy and the like) group may contain one ormore substituents. Examples of suitable substituents on the unsaturatedcarbon atom of an aryl or heteroaryl group include -halo, —NO₂, —CN,—R*, —C(R*)═C(R*)₂, —C≡C—R*, —OR*, —SR^(◯), —S(O)R^(◯), —SO₂R^(◯),—SO₃R*, —SO₂N(R⁺)₂, —N(R⁺)₂, —NR⁺C(O)R*, —NR⁺C(O)N(R⁺)₂,—N(R⁺)C(═NR⁺)—N(R⁺)₂, —N(R⁺)C(═NR⁺)—R^(◯), —NR⁺CO₂R^(◯), —NR⁺SO₂R^(◯),—NR⁺SO₂N(R⁺)₂, —O—C(O)R*, —O—CO₂R*, —OC(O)N(R⁺)₂, —C(O)R*, —CO₂R*,—C(O)—C(O)R*, —C(O)N(R⁺)₂, —C(O)N(R⁺)—OR*, —C(O)N(R⁺)C(═NR⁺)—N(R⁺)₂,—N(R⁺)C(═NR⁺)—N(R⁺)—C(O)R*, —C(═NR⁺)—N(R⁺)₂, —C(═NR⁺)—OR*,—N(R⁺)—N(R⁺)₂, —C(═NR⁺)—N(R⁺)—OR*, —C(R^(◯))═N—OR*, —P(O)(R*)₂,—P(O)(OR*)₂, —O—P(O)—OR*, and —P(O)(NR⁺)—N(R⁺)₂, wherein R^(◯), R⁺, andR* are as defined above, or two adjacent substituents, taken togetherwith their intervening atoms, form a 5-6 membered unsaturated orpartially unsaturated ring having 0-3 ring atoms selected from the groupconsisting of N, O, and S.

An aliphatic group or a non-aromatic heterocyclic ring may besubstituted with one or more substituents. Examples of suitablesubstituents on the saturated carbon of an aliphatic group or of anon-aromatic heterocyclic ring include, without limitation, those listedabove for the unsaturated carbon of an aryl or heteroaryl group and thefollowing: ═O, ═S, ═C(R*)₂, ═N—N(R*)₂, ═N—OR*, ═N—NHC(O)R*,═N—NHCO₂R^(◯), ═N—NHSO₂R^(◯), or ═N—R*, where each R* and R^(◯) is asdefined above.

Suitable substituents on a substitutable nitrogen atom of a heteroarylor non-aromatic heterocyclic ring include —R*, —N(R*)₂, —C(O)R*, —CO₂R*,—C(O)—C(O)R*—C(O)CH₂C(O)R*, —SO₂R*, —SO₂N(R*)₂, —C(═S)N(R*)₂,—C(═NH)—N(R*)₂, and —NR*SO₂R*; wherein each R* is as defined above. Aring nitrogen atom of a heteroaryl or non-aromatic heterocyclic ringalso may be oxidized to form the corresponding N-hydroxy or N-oxidecompound. A nonlimiting example of such a heteroaryl having an oxidizedring nitrogen atom is N-oxidopyridyl.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10%.

As used herein, the term “comprises” means “includes, but is not limitedto.”

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention. Unlessotherwise stated, structures depicted herein are also meant to includeall geometric (or conformational) isomers, i.e., (Z) and (E) double bondisomers and (Z) and (E) conformational isomers, as well as allstereochemical forms of the structure; i.e., the R and S configurationsfor each asymmetric center. Therefore, single stereochemical isomers aswell as enantiomeric and diasteromeric mixtures of the present compoundsare within the scope of the invention. When a mixture is enriched in onestereoisomer relative to another stereoisomer, the mixture may contain,for example, an enantiomeric excess of at least 50%, 75%, 90%, 99%, or99.5%.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructure except for the replacement of a hydrogen atom by a deuteriumor tritium, or the replacement of a carbon atom by a ¹³C- or¹⁴C-enriched carbon are within the scope of the invention.

As used herein, the term “seeding” is used to refer to the addition of acrystalline material to initiate crystallization or recrystallization.

When a compound crystallizes from a solution or slurry, it maycrystallize with different spatial lattice arrangements, a propertyreferred to as “polymorphism.” Each of the crystal forms is a“polymorph.” While polymorphs of a given substance have the samechemical composition, they may differ from each other with respect toone or more physical properties, such as solubility and dissociation,true density, melting point, crystal shape, compaction behavior, flowproperties, and/or solid state stability.

As used herein, the term “solvate or solvated” means a physicalassociation of a compound with one or more solvent molecules. Thisphysical association includes hydrogen bonding. In certain instances thesolvate will be capable of isolation, for example when one or moresolvent molecules are incorporated in the crystal lattice of thecrystalline solid. “Solvate or solvated” encompasses both solution-phaseand isolable solvates. Representative solvates include, for example,hydrates, ethanolates, or methanolates. The physical properties of asolvate typically differ from other solvates, and from unsolvated formsof the compound. Because the chemical composition also differs betweensolvates these forms are referred to as “pseudo-polymorphs”.

As used herein, the term “hydrate” is a solvate wherein the solventmolecule is H₂O that is present in a defined stoichiometric amount, andmay, for example, include hemihydrate, monohydrate, dihydrate, ortrihydrate. As used herein, the term “anhydrate” is a compound of theinvention that contains no H₂O incorporated in its crystal lattice.

As used herein, “crystalline” refers to a solid having a highly regularchemical structure. In particular, a crystalline compound may beproduced as one or more single crystalline forms of the compound. Forthe purposes of this application, the terms “single crystalline form” or“crystalline form” are used interchangeably and distinguish betweencrystals that have different properties (e.g., different XRPD patterns,different DSC scan results). Thus, each distinct polymorph andpseudopolymorph of a compound is considered to be a distinct singlecrystalline form herein.

“Substantially crystalline” refers to a compound that may be at least aparticular weight percent crystalline. Particular weight percentages are10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or anypercentage between 10% and 100%. In some embodiments, substantiallycrystalline refers to compounds that are at least 70% crystalline. Inother embodiments, substantially crystalline refers to compounds thatare at least 90% crystalline.

“Substantially pure” refers to a compound that may be at least aparticular weight percent of the compound. Particular weight percentagesare about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, orabout 99.5%.

Unless otherwise explicitly stated, structures depicted herein are meantto include all hydrates, anhydrates, solvates and polymorphs thereof.

As used herein, the terms “compound (I-1)” and“4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid” are used interchangeably, and include all crystalline forms. Bothterms refer to the compounds produced in Example 1 and Example 1A in theExamples below including both Form 1 and Form 2.

As used herein, the terms “compound (I-1) Form 2” and“4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid Form 2” are used interchangeably. Both terms refer to thecrystalline form 2 produced in Example 1 Form 2 and Example 1A in theExamples below.

As used herein, the terms “compound of formula (VIII-1)”, and“(R)-1-((2,5-dichlorobenzamido)acetamido)-3-methylbutylboronic acid” areused interchangeably. The compound of formula (VIII-1) is disclosed inU.S. Pat. No. 7,442,830 and WO 09/020448.

As used herein, the terms “compound of formula (I-15)”, “compound(I-15)” and “(I-15)” are used interchangeably and are used to refer tothe citrate ester of the compound (VIII-15), and the compound producedin Example 15 of the Examples below.

As used herein, the term “anhydride” used in reference to a boronic acidsuch as the compound of formula (VIII), refers to a chemical compoundformed by combination of two or more molecules of a boronic acidcompound, with loss of one or more water molecules. When mixed withwater, the boronic acid anhydride compound is hydrated to release thefree boronic acid compound. In various embodiments, the boronic acidanhydride can comprise two, three, four, or more boronic acid units, andcan have a cyclic or linear configuration. Non-limiting examples ofoligomeric boronic acid anhydrides of peptide boronic acids compound ofthe invention are illustrated below:

In formula (1) and (2), the variable nn is an integer from 0 to about10, preferably 0, 1, 2, 3, or 4. In some embodiments, the boronic acidanhydride compound comprises a cyclic trimer (“boroxine”) of formula(2), wherein an is 1. The variable W has the formula (3):

wherein P, R^(a2), A, R^(a1) and R^(a) are as defined herein.

As used herein, the total weight of a single oral pharmaceutical dosageform is determined by adding all the weights of the components in theoral pharmaceutical dosage form, and does not include the weight of anycoatings which may be optionally applied to the oral pharmaceuticaldosage form after it is formed. The total weight of a single oralpharmaceutical dosage form is used as the basis for calculating theweight percentage of each of the components that comprise the oralpharmaceutical dosage form.

As used herein, “low-moisture” used in reference to an excipient such asa filler, refers to an excipient that has a water content of about 0.5%to about 4%. The term “low-moisture” may be used interchangeably withthe term “low-water”.

As used herein, the term “lyophilized powder”, “cake”, or “lyophilizedcake” refers to any solid material obtained by lyophilization of anaqueous mixture.

As used herein, the term “tonicity modifier” refers to agents whichcontribute to the osmolality of a liquid or solution.

As used herein, the terms “boronate ester” and “boronic ester” are usedinterchangeably and refer to a chemical compound containing a —B(Z¹)(Z²)moiety, wherein Z¹ and Z² together form a moiety where the atom attachedto boron in each case is an oxygen atom.

In some embodiments, the boronate ester moiety is a 5-membered ring. Insome other embodiments, the boronate ester moiety is a 6-membered ring.In some other embodiments, the boronate ester moiety is a mixture of a5-membered ring and a 6-membered ring.

As used herein, the term “alpha-hydroxy carboxylic acid” refers to acompound that contains a hydroxyl group directly attached to a carbonatom in an alpha position relative to a carboxylic acid group. As usedherein, the term “alpha-hydroxy carboxylic acid” is not intended to belimited to compounds having only one hydroxyl group and one carboxylicacid group.

As used herein, the term “beta-hydroxy carboxylic acid” refers to acompound that contains a hydroxyl group directly attached to a carbonatom in a beta position relative to a carboxylic acid group. As usedherein, the term “beta-hydroxy carboxylic acid” is not intended to belimited to compounds having only one hydroxyl group and one carboxylicacid group.

As used herein, the term “moiety derived from an alpha-hydroxycarboxylic acid” refers to a moiety formed by removing a hydrogen atomfrom a carboxylic acid within an alpha-hydroxy carboxylic acid and byremoving a hydrogen atom from a hydroxyl group directly attached to acarbon atom in an alpha position relative to the carboxylic acid group.As used herein, the term “moiety derived from a beta-hydroxy carboxylicacid” refers to a moiety formed by removing a hydrogen atom from acarboxylic acid within a beta-hydroxy carboxylic acid and by removing ahydrogen atom from a hydroxyl group directly attached to a carbon atomin a beta position relative to the carboxylic acid group.

DETAILED DESCRIPTION OF THE INVENTION

In some embodiments the alpha-hydroxy acid is characterized by formula(V):

-   -   wherein each of R^(b3) and R^(b4) independently is hydrogen,        —CO₂H, or a substituted or unsubstituted aliphatic, aryl,        heteroaryl or heterocyclyl group.

In some embodiments, each of R^(b3) and R^(b4) independently ishydrogen, C₁₋₆ aliphatic, or —(CH₂)_(p)—CO₂H, and p is 0, 1 or 2. Insome embodiments, each of R^(b3) and R^(b4) independently is hydrogen orC₁₋₆ aliphatic. In certain such embodiments, each of R^(b3) and R^(b4)independently is selected from the group consisting of hydrogen, methyl,ethyl, isopropyl, isobutyl, tert-butyl, and cyclohexyl. In some otherembodiments, each of R^(b3) and R^(b4) independently is hydrogen or—(CH₂)_(p)—CO₂H. In some such embodiments, p is 1. In certain otherembodiments, each of R^(b3) and R^(b4) independently is —(CH₂)_(p)—CO₂H.In certain such embodiments, p is 1.

In some embodiments, the alpha-hydroxy carboxylic acid is selected fromthe group consisting of glycolic acid, malic acid, hexahydromandelicacid, citric acid, 2-hydroxyisobutyric acid, mandelic acid, lactic acid,2-hydroxy-3,3-dimethylbutyric acid, 2-hydroxy-3-methylbutyric acid,2-hydroxyisocaproic acid, and benzilic acid. In some other embodiments,the alpha-hydroxy carboxylic acid is selected from the group consistingof glycolic acid, malic acid, hexahydromandelic acid, citric acid,2-hydroxyisobutyric acid, mandelic acid, lactic acid,2-hydroxy-3,3-dimethylbutyric acid, 2-hydroxy-3-methylbutyric acid,2-hydroxyisocaproic acid, tartaric acid, and benzilic acid. In certainembodiments, the alpha-hydroxy carboxylic acid is citric acid. Someother non-limiting examples of alpha-hydroxy carboxylic acids includeglucoheptonic acid, maltonic acid, lactobionic acid, and galactaricacid.

In some embodiments the beta-hydroxy acid is characterized by formula(VI):

-   -   wherein each of R^(b1) and R^(b2) independently is hydrogen,        —CO₂H, —OH, or a substituted or unsubstituted aliphatic, aryl,        heteroaryl or heterocyclyl group; each of R^(b3) and R^(b4)        independently is hydrogen, —CO₂H, or a substituted or        unsubstituted aliphatic, aryl, heteroaryl or heterocyclyl group;    -   or R^(b2) and R^(b4) are each independently hydrogen, and R^(b1)        and R^(b3), taken together with the carbon atoms to which they        are attached, form an unsubstituted or substituted 4- to        8-membered non-aromatic ring having 0-3 ring heteroatoms        selected from the group consisting of O, N, and S, wherein said        ring may optionally be fused to an unsubstituted or substituted        4- to 8-membered non-aromatic ring or 5- to 6-membered aromatic        ring having 0-3 ring heteroatoms selected from the group        consisting of O, N, and S;    -   or R^(b2) and R^(b4) are absent, and R^(b1) and R^(b3), taken        together with the carbon atoms to which they are attached, form        an unsubstituted or substituted 5- to 6-membered aromatic ring        having 0-3 ring heteroatoms selected from the group consisting        of O, N, and S, wherein said ring may optionally be fused to an        unsubstituted or substituted 4- to 8-membered non-aromatic ring,        or 5- to 6-membered aromatic ring having 0-3 ring heteroatoms        selected from the group consisting of O, N, and S.

In some embodiments each of R^(b1) and R^(b2) independently is hydrogen,C₁₋₆ aliphatic, —(CH₂)_(p)—OH, or —(CH₂)_(p)—C₂, and p is 0, 1 or 2. Insome such embodiments, each of R^(b1) and R^(b2) is hydrogen. In someother such embodiments, R^(b1) is —OH and R^(b2) is hydrogen.

In some embodiments each of R^(b3) and R^(b4) independently is hydrogen,C₁₋₆ aliphatic, or —(CH₂)_(p)—CO₂H, and p is 0, 1 or 2. In someembodiments, each of R^(b3) and R^(b4) independently is hydrogen or C₁₋₆aliphatic. In certain such embodiments, each of R^(b3) and R^(b4)independently is selected from the group consisting of hydrogen, methyl,ethyl, isopropyl, isobutyl, tert-butyl, and cyclohexyl. In certain otherembodiments, each of R^(b3) and R^(b4) independently is —(CH₂)_(p)—CO₂H,and p is 0 or 1.

The variable p is 0, 1, or 2. In some embodiments, p is 0 or 1. Incertain embodiments, p is 0. In other certain embodiments, p is 1.

In some embodiments, R^(b2) and R^(b4) are absent and R^(b1) and R^(b3)taken together with the carbon atoms to which they are attached, form asubstituted or unsubstituted phenyl ring.

In some embodiments, the beta-hydroxy carboxylic acid is selected fromthe group consisting of malic acid, citric acid, 3-hydroxybutyric acid,beta-hydroxyisovaleric acid, and salicylic acid. In some otherembodiments, the beta-hydroxy carboxylic acid is selected from the groupconsisting of malic acid, citric acid, 3-hydroxybutyric acid,beta-hydroxyisovaleric acid, tartaric acid, and salicylic acid. Incertain embodiments, the beta-hydroxy carboxylic acid is citric acid.Some other non-limiting examples of beta-hydroxy carboxylic acidsinclude glucoheptonic acid, maltonic acid, lactobionic acid, andgalactaric acid. Some other non-limiting examples of beta-hydroxycarboxylic acids include embonic acid, 1-hydroxy-2-naphthoic acid and3-hydroxy-2-naphthoic acid.

In some embodiments, the alpha-hydroxy acid or beta-hydroxy acid isselected from the group consisting of glycolic acid, malic acid,hexahydromandelic acid, 2-hydroxyisobutyric acid, citric acid, mandelicacid, lactic acid, 3-hydroxybutyric acid, beta-hydroxyisovaleric acid,2-hydroxy-3,3-dimethylbutyric acid, 2-hydroxy-3-methylbutyric acid,2-hydroxyisocaproic acid, tartaric acid, salicylic acid, and benzilicacid.

In some embodiments, compounds of general formula (I) are characterizedby formula (II):

-   -   wherein:    -   the variables P, A, R^(a), R^(a1), R^(a2), and a have the values        described below and the variables R^(b1), R^(b2), R^(b3) and        R^(b4) have the values described above.

In some embodiments, any one of R^(b1), R^(b2), R^(b3) and R^(b4) maycontain a functional group that can form a further bond with the boronatom. In certain embodiments, the functional group is a carboxylic acid.In other certain embodiments, the functional group is a hydroxyl group.

In some embodiments, wherein the alpha-hydroxy carboxylic acid orbeta-hydroxy carboxylic acid is citric acid, the compound of generalformula (I) is characterized by formula (II) or (IV):

-   -   or a mixture thereof wherein the variables P, A, R^(a), R^(a1),        and R^(a2) have the values described below.

In some other embodiments, wherein the alpha-hydroxy carboxylic acid orbeta-hydroxy carboxylic acid is citric acid, a further bond can beformed between the carboxylic acid in formula (III) or (IV) and theboron atom. Without being limited by any chemical bonding theory, insuch embodiments, the compound of general formula (I) may be representedby formula (IIIa) or (IVa):

-   -   or a mixture thereof wherein the variables P, A, R^(a), R^(a1),        and R^(a2) have the values described below.

It is recognized that, without being limited by any chemical bondingtheory, there are other representations that could be used to depictthis further bonding of the carboxylic acid with the boron atom informulas (IIIa) and (IV).

The following values are described for the variables in any of formulas(I), (II), (III), (IIIa), (IV), or (IVa).

The variable P is hydrogen or an amino-group-blocking moiety.Non-limiting examples of amino-group-blocking moieties can be found inP. G. M. Wuts and T. W. Greene, Greene's Protective Groups in OrganicSynthesis (4^(th) ed.), John Wiley & Sons, NJ (2007), and include, e.g.,acyl, sulfonyl, oxyacyl, and aminoacyl groups.

In some embodiments, P is R^(c)—C(O)—, R^(c)—O—C(O)—,R^(c)—N(R^(4c))—C(O)—, R^(c)—S(O)₂—, or R^(c)—N(R^(4c))—S(O)₂—, whereR^(c) is selected from the group consisting of C₁₋₆ aliphatic, C₁₋₆fluoroaliphatic, —R^(D), -T¹-R^(D), and -T¹-R^(2c), and the variablesT¹, R^(D), R^(2c), and R^(4c) have the values described below.

The variable R^(4c) is hydrogen, C₁₋₄ alkyl, C₁₋₄ fluoroalkyl, or C₆₋₁₀ar(C₁₋₄ alkyl), the aryl portion of which is substituted orunsubstituted. In some embodiments, R^(4c) is hydrogen or C₁₋₄ alkyl. Incertain embodiments, R^(4c) is hydrogen.

The variable T¹ is a C₁₋₆ alkylene chain substituted with 0-2independently selected R^(3a) or R^(3b), wherein the alkylene chainoptionally is interrupted by —C(R⁵)═C(R⁵)—, —C≡C—, or —O—. Each R^(3a)independently is selected from the group consisting of —F, —OH, —O(C₁₋₄alkyl), —CN, —N(R⁴)₂, —C(O)—(C₁₋₄ alkyl), —CO₂H, —CO₂(C₁₋₄ alkyl),—C(O)NH₂, and —C(O)—NH(C₁₋₄ alkyl). Each R^(3b) independently is a C₁₋₃aliphatic optionally substituted with R^(3a) or R⁷; or two substituentsR^(3b) on the same carbon atom, taken together with the carbon atom towhich they are attached, form a 3- to 6-membered cycloaliphatic ring.Each R⁷ is a substituted or unsubstituted aromatic group. In someembodiments, T¹ is a C₁₋₄ alkylene chain.

The variable R^(2c) is halo, —OR⁵, —SR⁶, —S(O)R⁶, —SO₂R⁶, —SO₂N(R⁴)₂,—N(R⁴)₂, —NR⁴C(O)R⁵, —NR⁴C(O)N(R⁴)₂, —NR⁴CO₂R⁶, —N(R⁴)SO₂R⁶,—N(R⁴)SO₂N(R⁴)₂, —O—C(O)R⁵, —OC(O)N(R⁴)₂, —C(O)R⁵, —CO₂R⁵, or—C(O)N(R⁴)₂, where:

each R⁴ independently is hydrogen or an optionally substitutedaliphatic, aryl, heteroaryl, or heterocyclyl group; or two R⁴ on thesame nitrogen atom, taken together with the nitrogen atom, form anoptionally substituted 4- to 8-membered heterocyclyl ring having, inaddition to the nitrogen atom, 0-2 ring heteroatoms independentlyselected from the group consisting of N, O, and S;

each R⁵ independently is hydrogen or an optionally substitutedaliphatic, aryl, heteroaryl, or heterocyclyl group; and

each R⁶ independently is an optionally substituted aliphatic, aryl, orheteroaryl group.

The variable R^(D) is a substituted or unsubstituted aromatic,heterocyclyl, or cycloaliphatic ring, any of which is optionally fusedto a substituted or unsubstituted aromatic, heterocyclyl orcycloaliphatic ring. In some embodiments, R^(D) is substituted onsubstitutable ring carbon atoms with 0-2 R^(d) and 0-2 R^(8d), and eachsubstitutable ring nitrogen atom in R^(D) is unsubstituted or issubstituted with —C(O)R⁵, —C(O)N(R⁴)₂, —CO₂R⁶, —SO₂R⁶, —SO₂N(R⁴)₂, C₁₋₄aliphatic, a substituted or unsubstituted C₆₋₁₀ aryl, or a C₆₋₁₀ar(C₁₋₄)alkyl, the aryl portion of which is substituted orunsubstituted. The variables R⁴, R⁵, and R⁶ have the values describedabove. Each R^(d) independently is selected from the group consisting ofC₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic, halo, —R^(1d), —R^(2d), T²-R^(1d),and -T²-R^(2d), where the variables T², R^(1d), R^(2d), and R^(8d) havethe values described below. In some embodiments, each R^(d)independently is selected from the group consisting of C₁₋₆ aliphatic,C₁₋₆ fluoroaliphatic and halo.

T² is a C₁₋₆ alkylene chain substituted with 0-2 independently selectedR^(3a) or R^(3b), wherein the alkylene chain optionally is interruptedby —C(R⁵)═C(R⁵)—, —C≡C—, or —O—. The variables R^(3a), R^(3b), and R⁵have the values described above.

Each R^(1d) independently is a substituted or unsubstituted aryl,heteroaryl, heterocyclyl, or cycloaliphatic ring.

Each R^(2d) independently is —NO₂, —CN, —C(R⁵)═C(R)₂, —C≡C—R⁵, —OR⁵,—SR⁶, —S(O)R⁶, —SO₂R⁶, —SO₂N(R⁴)₂, —N(R⁴)₂, —NR⁴C(O)R⁵, —NR⁴C(O)N(R⁴)₂,—N(R⁴)C(═NR⁴)—N(R⁴)₂, —N(R⁴)C(═NR⁴)—R⁶, —NR⁴CO₂R⁶, —N(R⁴)SO₂R⁶,—N(R⁴)SO₂N(R⁴)₂, —O—C(O)R⁵, —OC(O)N(R⁴)₂, —C(O)R⁵, —CO₂R⁵, —C(O)N(R⁴)₂,—C(O)N(R⁴)—OR⁵, —C(O)N(R⁴)C(═NR⁴)—N(R⁴)₂, —N(R⁴)C(═NR⁴)—N(R⁴)—C(O)R⁵, or—C(═NR⁴)—N(R⁴)₂. The variables R⁴, R⁵, and R⁶ have the values describedabove.

Each R^(8d) independently is selected from the group consisting of C₁₋₄aliphatic, C₁₋₄ fluoroaliphatic, halo, —OH, —O(C₁₋₄ aliphatic), —NH₂,—NH(C₁₋₄ aliphatic), and —N(C₁₋₄ aliphatic)₂. In some embodiments, eachR^(8d) independently is C₁₋₄ aliphatic, C₁₋₄ fluoroaliphatic or halo.

In some embodiments, R^(D) is a substituted or unsubstituted mono- orbicyclic ring system. In some embodiments R^(D) is a substituted orunsubstituted mono- or bicyclic ring system selected from the groupconsisting of furanyl, thienyl, pyrrolyl, isoxazolyl, oxazolyl,thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl,thiadiazolyl, phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl,benzofuranyl, benzothiophenyl, indolyl, benzoxazolyl, benzisoxazolyl,benzimidazolyl, indazolyl, purinyl, naphthyl, quinolinyl, isoquinolinyl,cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, naphthyridinyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, tetrahydroquinoxalinyl,and dihydrobenzoxazinyl. In some embodiments, R^(D) is a substituted orunsubstituted mono- or bicyclic ring system selected from the groupconsisting of phenyl, pyridinyl, pyrimidinyl, pyrazinyl, naphthyl,benzimidazolyl, quinolinyl, isoquinolinyl, quinoxalinyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, tetrahydroquinoxalinyl,and dihydrobenzoxazinyl.

In some embodiments, the substitutable ring carbon atoms in R^(D) aresubstituted on substitutable carbon atoms with 0-1 R^(d) and 0-2 R^(8d);wherein:

each R^(d) independently is C₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic orhalo; and

each R^(8d) independently is C₁₋₄ aliphatic, C₁₋₄ fluoroaliphatic orhalo.

In some embodiments, the substitutable ring carbon atoms in R^(D) aresubstituted with 0-1 R^(d) and 0-2 R^(8d), wherein:

T¹ is a C₁₋₃ alkylene chain that is unsubstituted or is substituted withR^(3a) or R^(3b);

each R^(1d) independently is a substituted or unsubstituted aryl,heteroaryl, heterocyclyl, or cycloaliphatic ring; and

each R^(2d) independently is —OR⁵, —SR⁶, —S(O)R⁶, —SO₂R⁶, —SO₂N(R⁴)₂,—N(R⁴)₂, —NR⁴C(O)R⁵, —NR⁴C(O)N(R⁴)₂, —O—C(O)R⁵, —OC(O)N(R⁴)₂, —C(O)R⁵,—CO₂R⁵, or —C(O)N(R⁴)₂. The variables R⁴, R⁵, and R⁶ have the valuesdescribed above.

In some embodiments, the variable R^(d) has the formula -Q-R^(E), whereQ is —O—, —NH—, or —CH—, and R^(E) is a substituted or unsubstitutedaryl, heteroaryl, heterocyclyl, or cycloaliphatic ring. In someembodiments, R^(E) is a substituted or unsubstituted phenyl, pyridinyl,pyrimidinyl, pyrazinyl, piperidinyl, piperazinyl, or morpholinyl ring.

In some embodiments, P has the formula R^(c)—C(O)—, where R^(c) is C₁₋₄alkyl, C₁₋₄ fluoroalkyl, or C₆₋₁₀ ar(C₁₋₄) alkyl, the aryl portion ofwhich is substituted or unsubstituted. In certain such embodiments, P isselected from the group consisting of acetyl, trifluoroacetyl, andphenylacetyl.

In some other embodiments, P has the formula R^(D)—C(O)—, where R^(D) isa substituted or unsubstituted phenyl, pyridinyl, pyrazinyl,pyrimidinyl, quinolinyl, or quinoxalinyl. In yet some other embodiments,P has the formula R^(D)—C(O), where R^(D) is a phenyl, pyridinyl,pyrazinyl, pyrimidinyl, naphthyl, quinolinyl, quinoxalinyl,benzimidazolyl, or dihydrobenzoxazinyl substituted with 0-1 R^(d) and0-2 R^(8d).

In certain embodiments, P has the formula R^(D)—C(O)—, where R^(D) is2-pyrazinyl. In other certain embodiments, P has the formulaR^(D)—C(O)—, where R^(D) is 2,5-dichlorophenyl. In yet other certainembodiments, P has the formula R^(D)—C(O)—, where R^(D) is6-phenyl-2-pyridinyl.

In some other embodiments, P has the formula R^(c)—SO₂—, where R is—R^(D) or -T¹-R^(D), where T¹ is C₁₋₄ alkylene and R^(D) is a phenyl,pyridinyl, pyrazinyl, pyrimidinyl, naphthyl, quinolinyl, quinoxalinyl,benzimidazolyl, or dihydrobenzoxazinyl substituted with 0-1 R^(d) and0-2 R^(8d).

The variable R^(a) is hydrogen, C₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic,—(CH₂)_(m)—CH₂—R^(B), —(CH₂)_(m)—CH₂—NHC(═NR⁴)NH—Y,—(CH₂)_(m)—CH₂—CON(R⁴)₂, —(CH₂)_(m)—CH₂—N(R⁴)CON(R⁴)₂,—(CH₂)_(m)—CH(R⁶)N(R⁴)₂, —(CH₂)_(m)—CH(R^(5a))—OR^(5b), or—(CH₂)_(m)—CH(R⁵)—SR⁵, where the variables R⁴, R⁵, and R⁶ have thevalues described above, and the variables R^(5a), R^(5b), R^(B), Y, andm have the values described below.

In some embodiments, R^(a) is hydrogen, C₁₋₆ aliphatic, C₁₋₆fluoroaliphatic, or —(CH₂)_(m)—CH₂—R^(B). In some other embodiments,R^(a) is C₁₋₆ aliphatic, or —(CH₂)_(m)—CH₂—R^(B). In some furtherembodiments, R^(a) is C₁₋₆ aliphatic. In yet other further embodiments,R^(a) is isobutyl, l-naphthylmethyl, 2-naphthylmethyl, benzyl,4-fluorobenzyl, 4-hydroxybenzyl, 4-(benzyloxy)benzyl,benzylnaphthylmethyl or phenethyl. In certain embodiments, R^(a) isisobutyl.

The variable R^(a1) is hydrogen, C₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic,—(CH₂)_(m)—CH₂—R^(B), —(CH₂)_(m)—CH₂—NHC(═NR⁴)NH—Y,—(CH₂)_(m)—CH₂—CON(R⁴)₂, —(CH₂)_(m)—CH₂—N(R⁴)CON(R⁴)₂,—(CH₂)_(m)—CH(R⁶)N(R⁴)₂, —(CH₂)_(m)—CH(R^(5a))—OR^(5b), or—(CH₂)_(m)—CH(R⁵)—SR⁵, where the variables R⁴, R⁵, and R⁶ have thevalues described above, and the variables R^(5a), R^(5b), R^(B), Y, andm have the values described below.

In some embodiments, R^(a1) is hydrogen, C₁₋₆ aliphatic, C₁₋₆fluoroaliphatic, —(CH₂)_(m)—CH₂—R^(B), or —(CH₂)_(m)—CH(R^(5a))—OR^(5b).In some other embodiments, R^(a1) is hydrogen, —(CH₂)_(m)—CH₂—R^(B), or—(CH₂)_(m)—CH(R^(5a))—OR^(5b). In yet some other embodiments, R^(a1) isisobutyl, 1-naphthylmethyl, 2-naphthylmethyl, benzyl, 4-fluorobenzyl,4-hydroxybenzyl, 4-(benzyloxy)benzyl, benzylnaphthylmethyl or phenethyl.

In certain embodiments, R^(a1) is —CH₂—R^(B). In other certainembodiments, R^(a1) is —CH(R^(5a))—OR^(5b). In yet other certainembodiments, R^(a1) is hydrogen.

The variable R^(a2) is hydrogen, C₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic,—(CH₂)_(m)—CH₂—R^(B), —(CH₂)_(m)—CH₂—NHC(═NR⁴)NH—Y,—(CH₂)_(m)—CH₂—CON(R⁴)₂, —(CH₂)_(m)—CH₂—N(R⁴)CON(R⁴)₂,—(CH₂)_(m)—CH(R⁶)N(R⁴)₂, —(CH₂)_(m)—CH(R^(5a))—OR^(5b), or—(CH₂)_(m)—CH(R⁵)—SR⁵, where the variables R⁴, R⁵, and R⁶ have thevalues described above, and the variables R^(B), R^(5a), R^(5b), Y, andm have the values described below.

In some embodiments, R^(a2) is hydrogen, C₁₋₆ aliphatic, C₁₋₆fluoroaliphatic, —(CH₂)_(m)—CH₂—R^(B), or —(CH₂)_(m)—CH(R^(5a))—OR^(5b).In some other embodiments, R^(a2) is isobutyl, 1-naphthylmethyl,2-naphthylmethyl, benzyl, 4-fluorobenzyl, 4-hydroxybenzyl,4-(benzyloxy)benzyl, benzylnaphthylmethyl or phenethyl.

Each R^(B), independently, is a substituted or unsubstituted mono- orbicyclic ring system. In some embodiments, each R^(B) independently is asubstituted or unsubstituted phenyl, pyridyl, indolyl, benzimidazolyl,naphthyl, quinolinyl, quinoxalinyl, or isoquinolinyl ring. In certainembodiments, R^(B) is a substituted or unsubstituted phenyl ring.

The variable Y is hydrogen, —CN, or —NO₂. In some embodiments, Y is—NO₂.

The variable R^(5a) is hydrogen or a substituted or unsubstitutedaliphatic, aryl, heteroaryl, or heterocyclyl group. In some embodiments,R^(5a) is hydrogen or a substituted or unsubstituted aliphatic group. Insome other embodiments, R^(5a) is hydrogen or C₁₋₆ aliphatic. In suchembodiments, R^(5a) is selected from the group consisting of hydrogen,methyl, ethyl, isopropyl and isobutyl. In certain such embodiments,R^(5a) is methyl.

The variable R^(5b) is hydrogen or a substituted or unsubstitutedaliphatic, aryl, heteroaryl, or heterocyclyl group. In some embodiments,R^(5b) is hydrogen or a substituted or unsubstituted aliphatic group. Insome other embodiments, R^(5b) is hydrogen or C₁₋₆ aliphatic. In suchembodiments, R^(5b) is selected from the group consisting of hydrogen,methyl, ethyl, isopropyl and isobutyl. In certain such embodiments,R^(5b) is hydrogen.

The variable m is 0, 1, or 2. In some embodiments, m is 0 or 1. Incertain embodiments, m is 0. In other certain embodiments, m is 1.

The variable A is 0, 1, or 2. In some embodiments, A is 0 or 1. Incertain embodiments, A is 0.

The variable n is 0, or 1. In certain embodiments, n is 0. In othercertain embodiments, n is 1.

In some embodiments, A is 0; R^(a) is hydrogen, C₁₋₆ aliphatic, C₁₋₆fluoroaliphatic, or —(CH₂)_(m)—CH₂—R^(B); R^(a1) is hydrogen, C₁₋₆aliphatic, C₁₋₆ fluoroaliphatic, —(CH₂)_(m)—CH₂—R^(B), or—(CH₂)_(m)—CH(R^(5a))—OR^(5b); P is R^(c)—C(O)— or R^(c)—S(O)₂—; R^(c)is —R^(D); and m is 0 or 1.

In some other embodiments, A is 0; R^(a) is C₁₋₆ aliphatic or—(CH₂)_(m)—CH₂—R^(B); R^(a1) is hydrogen, —(CH₂)_(m)—CH₂—R^(B), or—(CH₂)_(m)—CH(R^(5a))—OR^(5b); P is R^(c)—C(O)— or R^(c)—S(O)₂—; R^(c)is —R^(D); and m is 0 or 1.

In some other embodiments, A is 0; R^(a) is C₁₋₆ aliphatic; R^(a1) ishydrogen, —(CH₂)_(m)—CH₂—R^(B), or —(CH₂)_(m)—CH(R^(5a))—OR^(5b); P isR^(c)—C(O); R^(c) is —R^(D); and m is 0 or 1.

In some other embodiments, A is 0; R^(a) is isobutyl; R^(a1) ishydrogen, C₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic, —(CH₂)_(m)—CH₂—R^(B), or—(CH₂)_(m)—CH(R^(5a))—OR^(5b); P is R^(c)—C(O)—; R^(c) is —R^(D); and mis 0 or 1.

In yet some other embodiments, A is 0; R^(a) is isobutyl; R^(a1) ishydrogen, C₁₋₆ aliphatic, —(CH₂)_(m)—CH₂—R^(B), or—(CH₂)_(m)—CH(R^(5a))—OR^(5b); P is R^(c)—C(O)—; R^(c) is —R^(D); and mis 0 or 1.

In still yet some other embodiments, A is 0; R^(a) is isobutyl; R^(a1)is hydrogen, —(CH₂)_(m)—CH₂—R^(B), or —(CH₂)_(m)—CH(R^(5a))—OR^(5b); Pis R^(c)—C(O)—; R^(c) is —R^(D); and m is 0 or 1.

In certain embodiments, A is 0; R^(a) is isobutyl; R^(a1) is —CH₂—R^(B),and R^(B) is phenyl; P is R^(c)—C(O)—; R^(c) is —R^(D); and R^(D) is2-pyrazinyl.

In other certain embodiments, A is 0; R^(a) is isobutyl; R^(a1) ishydrogen; P is R^(c)—C(O)—; R^(c) is —R^(D); and R^(D) is2,5-dichlorophenyl.

In yet other certain embodiments, A is 0; R^(a) is isobutyl; R^(a1) is—CH(R^(5a))—OR^(5b); R^(5a) is C₁₋₆ aliphatic; R^(5b) is hydrogen; P isR^(c)—C(O)—; R^(c) is —R^(D); and R^(D) is 6-phenyl-2-pyridinyl-.

In some embodiments, the compound of formula (I) is characterized byformula (I-1):

or a crystalline form thereof.

In some other embodiments, the compound of formula (I) is characterizedby formula (I-15):

or a crystalline form thereof.

In yet some other embodiments, the compound of formula (I) ischaracterized by formula (I-18):

or a crystalline form thereof.

General Synthetic Methodology

The compounds of formula (I) can be prepared by esterification ofcorresponding boronic acids. Such boronic acid compounds can be preparedfrom methods known to one of ordinary skill in the art. See, e.g., Adamset. al., U.S. Pat. No. 5,780,454; Pickersgill et al., InternationalPatent Publication WO 2005/097809. An exemplary synthetic route is setforth in Scheme 1 below.

Coupling of compound i with an N-protected amino acid ii, followed byN-terminal deprotection, provides compound iii or a salt thereof.Examples of suitable protecting groups (PG) include, without limitation,acyl protecting groups, e.g., formyl, acetyl (Ac), succinyl (Suc), andmethoxysuccinyl; and urethane protecting groups, e.g.,tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), andfluorenylmethoxycarbonyl (Fmoc). Optionally, PG is hydrogen anddeprotection is not necessary. The peptide coupling reaction can beconducted by prior conversion of the carboxylic acid moiety of compoundii to an activated ester or acid halide, e.g., anO—(N-hydroxysuccinimide) ester, followed by treatment with compound i.Alternatively, the activated ester can be generated in situ bycontacting the carboxylic acid with a peptide coupling reagent. Examplesof suitable peptide coupling reagents include, without limitation,carbodiimide reagents, e.g., dicyclohexylcarbodiimide (DCC) or1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC); phosphoniumreagents, e.g., (benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate (BOP); and uronium reagents, e.g.,O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU).

Compound ii is then coupled with an amino-group blocking moiety, toafford compound iv. The peptide coupling conditions described above forthe coupling of compounds i and ii are also suitable for couplingcompound iii with the amino-group blocking moiety. Deprotection of theboronic acid moiety then affords compound v. The deprotection steppreferably is accomplished by transesterification in a biphasic mixturecomprising the boronic ester compound iv, an organic boronic acidacceptor, a lower alkanol, a C₅₋₈ hydrocarbon solvent, and aqueousmineral acid. Other reagents that can be used for deprotection of theboronic acid moiety include, without limitation, BCl₃, lithium aluminiumhydride and NaIO₄.

Alternatively, the order of coupling reactions can be reversed, as shownin Scheme 2. Thus, an O-protected amino acid vi is first coupled with anamino-group blocking moiety, followed by ester hydrolysis, to formcompound vii. Optionally, PG′ is H and ester hydrolysis is not needed,leading directly to compound vii. Coupling with compound i and boronicacid deprotection are then accomplished as described above for Scheme 1to afford compound v.

Compound v is reacted with the appropriate alpha-hydroxy carboxylic acidor beta-hydroxy carboxylic acid to afford the compound of formula (I) asshown in Scheme 3.

The conversion of v to the compound of formula (I) can be accomplishedunder esterification conditions employing approximately a molarequivalent of the alpha-hydroxy carboxylic acid or beta-hydroxycarboxylic acid in a solvent such as ethyl acetate at a temperature ofbetween about 40° C. and about 80° C. The conversion of v to thecompound of formula (I) can also be accomplished as described aboveemploying a molar excess of the alpha-hydroxy carboxylic acid orbeta-hydroxy carboxylic acid. Examples of other suitable solvents forthis conversion include, but are not limited to, methyl isobutyl ketone,acetone, acetonitrile, 2-methyltetrahydrofuran, anisole, isopropylacetate, dimethoxyethane, tetrahydrofuran, dioxane, dichloromethane,toluene, heptane, methyl-cyclohexane, tert-butylmethyl ether, andmixtures thereof. The choice of the solvent will depend partly on thesolubility of the alpha-hydroxy carboxylic acid or beta-hydroxycarboxylic acid used. The temperature selected for the conversion of vto the compound of formula (I) will depend partly on the boiling pointof the solvent or solvent mixture used.

The conversion of v to the compound of formula (I) may be catalyzed byan organic amine base such as, but not limited to, triethylamine,triethylenediamine, pyridine, collidine, 2,6-lutidine,4-dimethylaminopyridine, di-tertbutylpyridine, N-methylmorpholine,N-methylpiperidine, tetramethylguanidine, diazabicyclo[5.4.0]undec-7-ene(DBU), 1,4-diazabicyclo[2.2.2]octane, 1,5-diazabicycle[4.3.0]non-5-ene,N,N′diisopropylethylamine, or a mixture thereof.

The compound of formula v and the alpha-hydroxy carboxylic acid orbeta-hydroxy carboxylic acid are heated together in the solvent ofchoice for a period of time. Following this period of time, the reactionmixture is allowed to cool for a period of time and the compound offormula (I) which precipitates upon cooling is collected by filtration.The cooling may be uncontrolled or may be controlled by the use of acooling apparatus. The reaction mixture may be stirred during thiscooling period. Alternatively, the compound of formula (I) can also beisolated from the reaction mixture by cooling followed by evaporation ofthe solvent. The reaction mixture may be seeded with crystals of thecompound of formula (I) in order to effect precipitation.

A co-solvent such as, but not limited to, heptane, methylcyclohexane,toluene, tert-butylmethyl ether, ethyl acetate, or a mixture thereof,may be added during the cooling period. Following the addition of theco-solvent, the reaction mixture can be cooled further leading to theprecipitation of the compound of formula (I). Alternatively, once theco-solvent is added, the reaction mixture can then be heated again togenerate a homogenous solution, which is then cooled leading to theprecipitation of the compound of formula (I). The reaction mixture maybe seeded with crystals of the compound of formula (I) in order toeffect precipitation.

In other embodiments, the compound of formula (I) is isolated insubstantially pure form. In such embodiments, the purity is about 80%,about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or about 99.5%.

In some embodiments, the compound of formula (I) is isolated incrystalline form. In some embodiments, the compound of formula (I) isisolated in substantially crystalline form. In some other embodiments,the compound of formula (I) is isolated in amorphous form.

The compound of formula (I) can also be generated by theco-lyophilization of compound v and the alpha-hydroxy carboxylic acid orbeta-hydroxy carboxylic acid. This is accomplished by subjecting anaqueous solution comprising the compound of formula v and a molar excessof the alpha-hydroxy carboxylic acid or beta-hydroxy carboxylic acid toa lyophilization procedure. In some embodiments, the aqueous solutionadditionally comprises a water-miscible co-solvent. Examples of suitableco-solvents include, but are not limited to, tert-butyl alcohol,methanol, ethanol, and mixtures thereof. The co-lyophilization resultsin a composition that contains the compound of formula (I) and theexcess alpha-hydroxy carboxylic acid or beta-hydroxy carboxylic acid.

Uses, Formulation, and Administration

The present invention provides compounds that are potent inhibitors ofthe proteasome. The compounds can be assayed in vitro or in vivo fortheir ability to inhibit proteasome-mediated peptide hydrolysis orprotein degradation.

In another aspect, therefore, the invention provides a method forinhibiting one or more peptidase activities of a proteasome in a cell,comprising contacting a cell in which proteasome inhibition is desiredwith a compound described herein, or a pharmaceutically acceptable salt,boronic ester, or boronic acid anhydride thereof.

The invention also provides a method for inhibiting cell proliferation,comprising contacting a cell in which such inhibition is desired with acompound described herein. The phrase “inhibiting cell proliferation” isused to denote the ability of a compound of the invention to inhibitcell number or cell growth in contacted cells as compared to cells notcontacted with the inhibitor. An assessment of cell proliferation can bemade by counting cells using a cell counter or by an assay of cellviability, e.g., an MTT or WST assay. Where the cells are in a solidgrowth (e.g., a solid tumor or organ), such an assessment of cellproliferation can be made by measuring the growth, e.g., with calipers,and comparing the size of the growth of contacted cells withnon-contacted cells.

Preferably, the growth of cells contacted with the inhibitor is retardedby at least about 50% as compared to growth of non-contacted cells. Invarious embodiments, cell proliferation of contacted cells is inhibitedby at least about 75%, at least about 90%, or at least about 95% ascompared to non-contacted cells. In some embodiments, the phrase“inhibiting cell proliferation” includes a reduction in the number ofcontacted cells, as compared to non-contacted cells. Thus, a proteasomeinhibitor that inhibits cell proliferation in a contacted cell mayinduce the contacted cell to undergo growth retardation, to undergogrowth arrest, to undergo programmed cell death (i.e., apoptosis), or toundergo necrotic cell death.

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of formula (I), or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable carrier.

In some embodiments, the composition also comprises the freealpha-hydroxy carboxylic acid or a salt thereof or the beta-hydroxycarboxylic acid or a salt thereof. In such embodiments, thealpha-hydroxy carboxylic acid or a salt thereof or beta-hydroxycarboxylic acid or a salt thereof and the compound of formula (I) arepresent in a molar ratio ranging from about 2:1 to about 200:1. Invarious embodiments, the alpha hydroxy carboxylic acid or a salt thereofor beta-hydroxy carboxylic acid or a salt thereof and the compound offormula (I) are present in a ratio ranging from about 2:1 to about200:1, from about 15:1 to about 80:1, or from about 20:1 to about 40:1.

If a pharmaceutically acceptable salt of the compound of the inventionis utilized in these compositions, the salt preferably is derived froman inorganic or organic acid or base. For reviews of suitable salts,see, e.g., Berge et al, J. Pharm. Sci. 66:1-19 (1977) and Remington: TheScience and Practice of Pharmacy, 20th Ed., ed. A. Gennaro, LippincottWilliams & Wilkins, 2000.

Nonlimiting examples of suitable acid addition salts include thefollowing: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate,persulfate, 3-phenyl-propionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate and undecanoate.

Suitable base addition salts include, without limitation, ammoniumsalts, alkali metal salts, such as lithium, sodium and potassium salts;alkaline earth metal salts, such as calcium and magnesium salts; othermultivalent metal salts, such as zinc salts; salts with organic bases,such as dicyclohexylamine, N-methyl-D-glucamine, t-butylamine, ethylenediamine, ethanolamine, and choline; and salts with amino acids such asarginine, lysine, and so forth.

The term “pharmaceutically acceptable carrier” is used herein to referto a material that is compatible with a recipient subject, preferably amammal, more preferably a human, and is suitable for delivering anactive agent to the target site without terminating the activity of theagent. The toxicity or adverse effects, if any, associated with thecarrier preferably are commensurate with a reasonable risk/benefit ratiofor the intended use of the active agent.

The terms “carrier”, “excipient” or “vehicle” are used interchangeablyherein, and include any and all solvents, diluents, and other liquidvehicles, dispersion or suspension aids, surface active agents, pHmodifiers, isotonic agents, thickening or emulsifying agents,preservatives, solid binders, lubricants and the like, as suited to theparticular dosage form desired. Remington: The Science and Practice ofPharmacy, 20th Ed., ed. A. Gennaro, Lippincott Williams & Wilkins, 2000discloses various carriers used in formulating pharmaceuticallyacceptable compositions and known techniques for the preparationthereof. Strickley, Pharmaceutical Research, 21(2) 201-230 (2004)reviews pharmaceutically acceptable excipients used in commercialproducts to solubilize compounds for oral or parenteral administration.Except insofar as any conventional carrier medium is incompatible withthe compounds of the invention, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutically acceptable composition,its use is contemplated to be within the scope of this invention. Someexamples of materials which can serve as pharmaceutically acceptablecarriers include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, carbonates, magnesiumhydroxide and aluminum hydroxide, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, pyrogen-free water, salts or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, and zinc salts, colloidal silica, magnesiumtrisilicate, polyvinyl pyrrolidone, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, wool fat, sugars such aslactose, glucose, sucrose, and mannitol, starches such as corn starchand potato starch, cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate, powderedtragacanth; malt, gelatin, talc, excipients such as cocoa butter andsuppository waxes, oils such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil, glycols such aspropylene glycol and polyethylene glycol, esters such as ethyl oleateand ethyl laurate, agar, alginic acid, isotonic saline, Ringer'ssolution, alcohols such as ethanol, isopropyl alcohol, hexadecylalcohol, and glycerol, cyclodextrins such as hydroxypropylβ-cyclodextrin and sulfobutylether β-cyclodextrin, lubricants such assodium lauryl sulfate and magnesium stearate, petroleum hydrocarbonssuch as mineral oil and petrolatum. Coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The pharmaceutical compositions of the invention can be manufactured bymethods well known in the art such as conventional granulating, mixing,dissolving, encapsulating, lyophilizing, or emulsifying processes, amongothers. Compositions may be produced in various forms, includinggranules, precipitates, or particulates, powders, including freezedried, rotary dried or spray dried powders, amorphous powders, tablets,capsules, syrup, suppositories, injections, emulsions, elixirs,suspensions or solutions.

According to a preferred embodiment, the compositions of this inventionare formulated for pharmaceutical administration to a mammal, preferablya human being. Such pharmaceutical compositions of the present inventionmay be administered orally, parenterally, by inhalation spray,topically, rectally, nasally, buccally, vaginally or via an implantedreservoir. The term “parenteral” as used herein includes subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques. Preferably, the compositions areadministered orally, intravenously, or subcutaneously. The formulationsof the invention may be designed to be short-acting, fast-releasing, orlong-acting. Still further, compounds can be administered in a localrather than systemic means, such as administration (e.g., by injection)at a tumor site.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, cyclodextrins, dimethylformamide, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor, and sesameoils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols andfatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Theinjectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use. Compositions formulated for parenteral administration may beinjected by bolus injection or by timed push, or may be administered bycontinuous infusion.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, cellulose,sucrose, glucose, mannitol, and silicic acid, b) binders such as, forexample, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, crospovidone,cellulose, croscarmellose sodium, sodium starch glycolate, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, cetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, sodium stearyl fumarate,stearic acid, solid polyethylene glycols, sodium lauryl sulfate,glyceryl behenate, and mixtures thereof. In the case of capsules,tablets and pills, the dosage form may also comprise buffering agentssuch as phosphates or carbonates.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such asmagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes. In some embodiments,the excipients or carriers may include, but are not limited to sodiumstearyl fumarate, carboxymethylcellulose, magnesium stearate,crospovidone, ethylcellulose, talc, and silicified microcrystallinecellulose.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

In some embodiments, the present invention provides pharmaceuticalcompositions comprising the compound of formula (I), and additionalexcipients described herein. In some other embodiments, the presentinvention provides pharmaceutical compositions comprising the compoundof formula (II), and additional excipients described herein. In yet someother embodiments, the present invention provides pharmaceuticalcompositions comprising the compound of formula (III) or (IV), andadditional excipients described herein.

In further embodiments, the present invention provides pharmaceuticalcompositions comprising the citrate ester of compound (VIII-1), andadditional excipients described herein. In other further embodiments,the present invention provides pharmaceutical compositions comprisingthe citrate ester of compound (VIII-15), and additional excipientsdescribed herein. In further embodiments, the present invention providespharmaceutical compositions comprising the citrate ester of (VIII-18)and additional excipients described herein.

In further embodiments, the present invention provides pharmaceuticalcompositions comprising the compound (I-1), or a crystalline formthereof. In still yet further embodiments, the present inventionprovides pharmaceutical compositions comprising the compound (I-15), ora crystalline form thereof. In still yet some further embodiments, thepresent invention provides pharmaceutical compositions comprising thecompound (I-18), or a crystalline form thereof.

The following description of pharmaceutical compositions and methods forpreparation of said pharmaceutical compositions are applicable to thecompounds of formulas (I), (II), (III), (IIIa), (IV), or (IVa) andvarious embodiments of these formulas as described herein. The followingdescription of pharmaceutical compositions and methods for preparationof said pharmaceutical compositions are also applicable to the compounds(I-1), (I-15), or (I-18).

In one embodiment, the pharmaceutical composition comprises the compoundof formula (I), wherein the compound of formula (I) is substantiallycrystalline. In another embodiment, the compound of formula (I) in thepharmaceutical composition is at least about 95%, 96%, 97%, 98%, 99%,99.5%, 99.9% of a crystalline form. In yet another embodiment, thecompound of formula (I) in the pharmaceutical composition is acrystalline form.

In some embodiments, the pharmaceutical formulations of the inventionprovide stable solid oral dosage forms of active compound, by usingexcipients having low water or low-moisture content, and manufacturedusing dry or non-aqueous formulation processes.

In one embodiment, the pharmaceutical composition is an oralpharmaceutical dosage form, selected from the group consisting ofcapsules, tablets, pills, powders, and granules. In another embodiment,the oral pharmaceutical dosage form is a capsule, wherein the capsule isa polymer-based capsule selected from the group consisting of gelatin,hydroxypropylmethyl cellulose (HPMC), fish gelatin, and pullulan. In yetanother embodiment, the polymer-based capsule is selected from the groupconsisting of gelatin, and hydroxypropylmethyl cellulose. In still yetanother embodiment, the polymer-based capsule is a hard gelatin capsule.

In one embodiment, the pharmaceutical composition comprises the compoundof formula (I), or a crystalline form thereof, a filler, and optionallya lubricant. In another embodiment, the pharmaceutical compositioncomprises about 0.2% to about 3% of the compound of formula (I), or acrystalline form thereof; about 97% to about 99.8% of a filler, andoptionally up to about 1.5% of a lubricant. In yet another embodiment,the pharmaceutical composition comprises about 0.25% to about 2% of thecompound of formula (I), or a crystalline form thereof; and about 98% toabout 99.75% of a filler.

In another embodiment, the pharmaceutical composition further comprisesan optional flow-aid, and an optional buffer. In yet another embodiment,the pharmaceutical composition comprises about 0.2% to about 3% of thecompound of formula (I), or a crystalline form thereof, about 86.5% toabout 99.8% of a filler, optionally up to about 1.5% of a lubricant,optionally up to about 5% of a flow-aid, and optionally up to about 5%of a buffer, by weight as a percentage of total weight.

In another embodiment, the pharmaceutical composition comprises about0.2% to about 12% of the compound of formula (I), or a crystalline formthereof, about 76.5% to about 99.8% of a filler, optionally up to about1.5% of a lubricant, optionally up to about 5% of a flow-aid, andoptionally up to about 5% of a buffer, by weight as a percentage oftotal weight.

In some embodiments, the compound of formula (I), or a crystalline formthereof, is present in the pharmaceutical composition in an amount ofabout 0.2% to about 3%, by weight as a percentage of total weight. Insome other embodiments, the compound of formula (I), or a crystallineform thereof is present in the pharmaceutical composition, in an amountof about 0.25% to about 2%, by weight as a percentage of total weight.

Suitable fillers include, but are not limited to, powdered cellulose,microcrystalline cellulose, silicified microcrystalline cellulose, highdensity microcrystalline cellulose, low-moisture microcrystallinecellulose, pregeletanized starch, sodium starch glycolate, and mixturesthereof. In some other embodiments, the filler is selected from thegroup consisting of powdered cellulose, microcrystalline cellulose,silicified microcrystalline cellulose, low-moisture microcrystallinecellulose, and mixtures thereof. In yet some other embodiments, thefiller is low-moisture microcrystalline cellulose. In some furtherembodiments, the filler is selected from the group consisting oflow-moisture microcrystalline cellulose, sodium starch glycolate,pregeletanized starch, and mixtures thereof.

In other embodiments, the filler is present in an amount of about 97% toabout 99.8%, by weight as a percentage of total weight. In some otherembodiments, the filler is present in an amount from about 98% to about99.75%, by weight as a percentage of total weight. In yet some otherembodiments, when a lubricant is present, the amount of filler isreduced by the corresponding percent amount of lubricant present. Insome further embodiments, the filler is present in an amount of about86.5% to about 99.8%, by weight as a percentage of total weight.

In some embodiments, the filler comprises a first filler and a secondfiller. The first filler is present in an amount of 0% to about 99.8%,by weight as a percentage of total weight, and the second filler ispresent in an amount of 0% to about 99.8% by weight as a percentage oftotal weight, as long as the total amount of filler is no greater thanabout 99.8%. In some embodiments, the first filler is present in anamount of about 40% to about 60%, by weight as a percentage of totalweight, and the second filler is present in an amount of about 40% toabout 60% by weight as a percentage of total weight, as long as thetotal amount of filler is not greater than about 99.8%, by weight as apercentage of total weight.

In some embodiments, the first filler is selected from the groupconsisting of low-moisture microcrystalline cellulose, sodium starchglycolate, pregeletanized starch, and mixtures thereof. In someembodiments, the second filler is selected from the group consisting oflow-moisture microcrystalline cellulose, sodium starch glycolate,pregeletanized starch, and mixtures thereof.

Suitable lubricants include, but are not limited to, magnesium stearate,glyceryl behenate, hydrogenated vegetable oil, talc, zinc stearate,calcium stearate, sucrose stearate, sodium stearyl fumarate, andmixtures thereof. In some embodiments, the lubricant is magnesiumstearate. In other embodiments, the lubricant is present in an amount ofup to about 1.5%, by weight as a percentage of total weight. In yet someother embodiments, the lubricant is present in an amount of about 1%, byweight as a percentage of total weight.

Suitable flow-aids include, but are not limited to silicon dioxide,talc, and mixtures thereof. In some embodiments, the flow-aid is talc.In other embodiments, the flow-aid is present in an amount of up toabout 5%, by weight as a percentage of total weight. In some otherembodiments, the flow-aid is present in an amount of about 1%, by weightas a percentage of total weight. In yet some other embodiments, theflow-aid is present in an amount of about 2%, by weight as a percentageof total weight.

Suitable buffers include sodium citrate, citric acid, and mixturesthereof. In some embodiments, the buffer is sodium citrate. In someother embodiments, the buffer is present in an amount of up to about 5%,by weight as a percentage of total weight. In yet some otherembodiments, the buffer is present in an amount of about 2%, by weightas a percentage of total weight.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I), or a crystalline form thereof a filler, andoptionally a lubricant; wherein:

-   -   the alpha-hydroxy carboxylic acid or beta-hydroxy carboxylic        acid is citric acid;    -   A is 0;    -   R^(a) is isobutyl;    -   R^(a1) is hydrogen, C₁₋₆ aliphatic, —(CH₂)_(m)—CH₂—R^(B), or        —(CH₂)_(m)—CH(R^(5a))—OR^(5b);    -   P is R^(c)—C(O)—;    -   R^(c) is —R^(D);    -   m is 0 or 1;    -   the filler is selected from the group consisting of low-moisture        microcrystalline cellulose, sodium starch glycolate,        pregeletanized starch, and mixtures thereof; and    -   the lubricant, when present, is magnesium stearate.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I), or a crystalline form thereof a filler, andoptionally a lubricant; wherein:

-   -   the compound of formula (I) is (I-1), (I-15) or (I-18);    -   the filler is selected from the group consisting of low-moisture        microcrystalline cellulose, sodium starch glycolate,        pregeletanized starch, and mixtures thereof; and    -   the lubricant, when present, is magnesium stearate.

In some embodiments, the pharmaceutical composition comprises about0.25% to about 2% of the compound of formula (I), or a crystalline formthereof; and about 98% to about 99.75% of a filler, wherein:

-   -   the compound of formula (I) is (I-1), (I-15) or (I-18); and    -   the filler is selected from the group consisting of low-moisture        microcrystalline cellulose, sodium starch glycolate,        pregeletanized starch, and mixtures thereof.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I), or a crystalline form thereof a filler,optionally a lubricant; optionally a flow-aid; and optionally a buffer,wherein:

-   -   the alpha-hydroxy carboxylic acid or beta-hydroxy carboxylic        acid is citric acid;    -   A is 0;    -   R^(a) is isobutyl;    -   R^(a1) is hydrogen, C₁₋₆ aliphatic, —(CH₂)_(m)—CH₂—R^(B), or        —(CH₂)_(m)—CH(R^(5a))—OR^(5b);    -   P is R^(c)—C(O)—;    -   R^(c) is —R^(D);    -   m is 0 or 1;    -   the filler is selected from the group consisting of low-moisture        microcrystalline cellulose, sodium starch glycolate,        pregeletanized starch, and mixtures thereof;    -   the lubricant, when present, is magnesium stearate;    -   the flow-aid, when present, is talc; and    -   the buffer, when present, is sodium citrate.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I), or a crystalline form thereof a filler,optionally a lubricant; optionally a flow-aid; and optionally a buffer,wherein:

-   -   the compound of formula (I) is (I-1), (I-15) or (I-18);    -   the filler is selected from the group consisting of low-moisture        microcrystalline cellulose, sodium starch glycolate,        pregeletanized starch, and mixtures thereof;    -   the lubricant, when present, is magnesium stearate;    -   the flow-aid, when present, is talc; and    -   the buffer, when present, is sodium citrate.

In some embodiments, the pharmaceutical composition comprises about 0.2%to about 3% of the compound of formula (I), or a crystalline formthereof, about 86.5% to about 99.8% of a filler, optionally up to about1.5% of a lubricant, optionally up to about 5% of a flow-aid, andoptionally up to about 5% of a buffer, by weight as a percentage oftotal weight, wherein:

-   -   the compound of formula (I) is (I-1), (I-15) or (I-18);    -   the filler is selected from the group consisting of low-moisture        microcrystalline cellulose, sodium starch glycolate,        pregeletanized starch, and mixtures thereof;    -   the lubricant, when present, is magnesium stearate;    -   the flow-aid, when present, is talc; and    -   the buffer, when present, is sodium citrate.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I), or a crystalline form thereof, a filler, andoptionally a lubricant; wherein the compound of formula (I) is (I-1). Insome other embodiments, the pharmaceutical composition comprises thecompound of formula (I), or a crystalline form thereof, a filler, andoptionally a lubricant; wherein the compound of formula (I) is (I-1);the filler is selected from the group consisting of low-moisturemicrocrystalline cellulose, sodium starch glycolate, pregeletanizedstarch, and mixtures thereof; and the lubricant, when present, ismagnesium stearate.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I), or a crystalline form thereof; wherein thecompound of formula (I) is (I-1); and the crystalline form is Form 2.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I-1) Form 2, and low-moisture microcrystallinecellulose. In some other embodiments, the pharmaceutical compositioncomprises the compound of formula (I-1) Form 2, and silicifiedmicrocrystalline cellulose. In yet some other embodiments, thepharmaceutical composition comprises the compound of formula (I-1) Form2, low-moisture microcrystalline cellulose, and magnesium stearate. Instill yet some further embodiments, the pharmaceutical compositioncomprises the compound of formula (I-1) Form 2, microcrystallinecellulose, and magnesium stearate.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I-1) Form 2, low-moisture microcrystallinecellulose, and talc. In some other embodiments, the pharmaceuticalcomposition comprises the compound of formula (I-1) Form 2, andpregeletanized starch. In yet some other embodiments, the pharmaceuticalcomposition comprises the compound of formula (I-1) Form 2,pregeletanized starch, talc, and magnesium stearate. In still yet someother embodiments, the pharmaceutical composition comprises the compoundof formula (I-1) Form 2, low-moisture microcrystalline cellulose, talc,and magnesium stearate. In some further embodiments, the pharmaceuticalcomposition comprises the compound of formula (I-1) Form 2, low-moisturemicrocrystalline cellulose, talc, magnesium stearate, and sodiumcitrate. In some other further embodiments, the pharmaceuticalcomposition comprises the compound of formula (I-1) Form 2, low-moisturemicrocrystalline cellulose, talc, magnesium stearate, and pregeletanizedstarch. In still yet some further embodiments, the pharmaceuticalcomposition comprises the compound of formula (I-1) Form 2, low-moisturemicrocrystalline cellulose, talc, magnesium stearate, and sodium starchglycolate.

When the compound of formula (I) is subjected to hydrolytic conditions,the ester portion of the molecule is hydrolyzed to give the compound offormula (VIII) in a 1:1 molecular ratio.

Using an analytical method that involves hydrolytic conditions forsample preparation, the amount of the compound of formula (VIII) presentin a test sample is measured (see e.g. Analytical Test Method 1, below),by comparison to a reference standard of known purity. Using ananalytical method that does not subject the sample to hydrolyticconditions, the amount of the compound of formula (VIII) present in thesample of the compound of formula (I) is measured by comparison to areference standard of known purity (see e.g. Analytical Test Method 2below). Therefore, the amount of the compound of formula (VIII) measuredin Analytical Test Method 1 minus the amount of the compound of formula(VIII) measured in Analytical Test Method 2, gives the amount of thecompound of formula (VIII) in the sample that is derived from hydrolysisof the compound of formula (I). Based on a 1:1 molecular ratio for theconversion of the compound of formula (I) to the compound of formula(VIII), a molecular weight conversion gives the amount of compound offormula (I) present in the test sample.

It will be recognized that such analytical methods as described directlyabove, and in the Experimental section below are applicable in a similarmanner to any of the compounds of formulas (I), (II), (III), (IIIa),(IV), or (IVa) and various embodiments of these formula as describedherein. Such analytical methods as described directly above and in theExperimental section below are applicable in a similar manner to thecompounds (I-1), (I-15), or (I-18).

In some embodiments, the amount of the compound of formula (VIII)present in a pharmaceutical composition is determined by measuring theamount of the compound of formula (VIII) that is present aftersubjecting to the sample to conditions under which the compound offormula (I) is hydrolyzed to the compound of formula (VIII).

In some embodiments, the amount of the compound of formula (I-1), or acrystalline form thereof, present in a pharmaceutical composition isexpressed as the equivalent amount on a molar weight basis of thecompound of formula (VIII-1).

In some embodiments, the invention relates to a unit dose pharmaceuticalcomposition comprising the compound of formula (I-1), or a crystallineform thereof.

In some other embodiments, the unit dose pharmaceutical compositioncomprises the compound of formula (I-1), or a crystalline form thereof,wherein the compound of formula (I-1) is present in an amount equivalentto a molar weight basis of about 0.1 mg to about 3.0 mg of the compoundof formula (VIII-1). In yet some other embodiments, the unit dosepharmaceutical composition comprises the compound of formula (I-1), or acrystalline form thereof, wherein the compound of formula (I-1) ispresent in an amount equivalent to a molar weight basis of about 0.15 mgto about 2.2 mg of the compound of formula (VIII-1). In still yet someother embodiments, the unit dose pharmaceutical composition comprisesthe compound of formula (I-1), or a crystalline form thereof, whereinthe compound of formula (I-1) is present in an amount equivalent to amolar weight basis of about 0.18 mg to about 0.22 mg of the compound offormula (VIII-1). In some further embodiments, the unit dosepharmaceutical composition comprises the compound of formula (I-1), or acrystalline form thereof, wherein the compound of formula (I-1) ispresent in an amount equivalent to a molar weight basis of about 0.46 mgto about 0.54 mg of the compound of formula (VIII-1). In still somefurther embodiments, the unit dose pharmaceutical composition comprisesthe compound of formula (I-1), or a crystalline form thereof, whereinthe compound of formula (I-1) is present in an amount equivalent to amolar weight basis of about 1.80 mg to about 2.20 mg of the compound offormula (VIII-1).

In some embodiments, the amount of the compound of formula (I-1), or acrystalline form thereof present in a pharmaceutical composition isexpressed as the equivalent amount of the compound of formula (VIII-1),based on the relative molecular weights of the compound of formula (I-1)and the compound of formula (VIII-1).

In some embodiments, the unit dose pharmaceutical composition comprisesabout 0.143 mg to about 4.3 mg of the compound of formula (I-1), or acrystalline form thereof measured as about 0.1 mg to about 3.0 mg of thecompound of formula (VIII-1), on a weight for weight basis.

In some other embodiments, the unit dose pharmaceutical compositioncomprises about 0.214 mg to about 3.15 mg of the compound of formula(I-1), or a crystalline form thereof measured as about 0.15 mg to about2.2 mg of the compound of formula (VIII-1), on a weight for weightbasis.

In yet some other embodiments, the unit dose pharmaceutical compositioncomprises about 0.258 mg to about 0.315 mg of the compound of formula(I-1), or a crystalline form thereof, measured as about 0.18 mg to about0.22 mg of the compound of formula (VIII-1), on a weight for weightbasis.

In still yet some other embodiments, the unit dose pharmaceuticalcomposition comprises about 0.659 mg to about 0.773 mg of the compoundof formula (I-1), or a crystalline form thereof measured as about 0.46mg to about 0.54 mg of the compound of formula (VIII-1), on a weight forweight basis.

In some further embodiments, the unit dose pharmaceutical compositioncomprises about 2.58 mg to about 3.15 mg of the compound of formula(I-1), or a crystalline form thereof, measured as about 1.80 mg to about2.20 mg of the compound of formula (VIII-1), on a weight for weightbasis.

In some embodiments, the invention provides a process for the productionof an oral pharmaceutical dosage form of the compound of formula (I), ora crystalline form thereof, wherein the oral pharmaceutical dosage formis a capsule, comprising the steps of:

-   -   (a-1) mixing together screened filler and screened compound of        formula (I), or a crystalline form thereof, in a bag;    -   (a-2) passing the resulting mixture from step (a-1) through a        screen, then blending;    -   (a-3) screening additional filler through the same screen,        passing it through the same bag, and blending in the same        blending apparatus;    -   (a-4) repeating step (a-3) up to two times;    -   (a-5) taking the resulting mixture from step (a-4), and        encapsulating it using a capsule-filling system; and    -   (a-6) weight-sorting the resulting capsules from step (a-5).

In some embodiments, step (a-3) may be repeated three or more times.

When a lubricant is present in the pharmaceutical composition, theinvention provides a process for the production of an oralpharmaceutical dosage form of the compound of formula (I), or acrystalline form thereof, wherein the oral pharmaceutical dosage form isa capsule, comprising the steps of:

-   -   (b-1) mixing together screened filler, and screened compound of        formula (I), or a crystalline form thereof, in a bag;    -   (b-2) passing the resulting mixture from step (b-1) through a        screen, then blending;    -   (b-3) screening additional filler through the same screen,        passing it through the same bag, and blending in the same        blending apparatus;    -   (b-4) repeating step (b-3) up to two times;    -   (b-5) blending together the mixture from step (b-4), and        screened lubricant;    -   (b-6) taking the resulting mixture from step (b-5), and        encapsulating it using a capsule-filling system; and    -   (b-7) weight-sorting the resulting capsules from step (b-6).

In some embodiments, step (b-3) may be repeated three or more times.When additional components are present in the pharmaceuticalcomposition, such as buffer, second filler, or flow-aid, they may beadded in any of steps (b-1) or (b-3). The total amount of each componentof the pharmaceutical composition may be added in one step or may bebroken into several amounts, which may or may not be of equal weight,and added in individual occurrences of steps (b-1) or (b-3).

In some embodiments, the invention provides a process for the productionof an oral pharmaceutical dosage form of the compound of formula (I), ora crystalline form thereof, wherein the oral pharmaceutical dosage formis a capsule, comprising the steps of:

-   -   (c-1) passing filler through a screen, then placing in a high        shear mixing apparatus;    -   (c-2) passing the compound of formula (I), or a crystalline form        thereof, through a screen, then placing in the same high shear        mixing apparatus;    -   (c-3) passing filler through a screen, then placing in the same        high shear mixing apparatus;    -   (c-4) mixing using the same high shear mixing apparatus for less        than 10 minutes;    -   (c-5) taking the resulting mixture from step (c-4), and        encapsulating it using a capsule-filling system; and    -   (c-6) weight-sorting the resulting capsules from step (c-5).

In some embodiments, when using the high shear mixing apparatus,additional components that are present in the pharmaceutical compositionmay be added by repeating either step (c-1) or step (c-3).

In some embodiments, the compound of formula (I) used in the processesfor preparation of solid oral dosage forms described above is selectedfrom the group consisting of (I-1), (I-15), and (I-18). In someembodiments, the compound of formula (I) used in the processes forpreparation of solid oral dosage forms described above is (I-1).

The process steps outlined above can take place using conventionalapparatus and equipment. For a review, see e.g. Remington: The Scienceand Practice of Pharmacy, 21^(st) Ed., Lippincott Williams & Wilkins,2005.

The blending steps outlined above can take place in any conventionalblending apparatus. In some embodiments, the blending time for eachindividual blending step is between about 1 minute and about 45 minutes.In some other embodiments, the blending time for each individualblending step is between about 1 minute and about 20 minutes. In yetsome other embodiments, the blending time for each individual blendingstep is between about 2 minutes and about 15 minutes.

The mixing step outlined above can take place in any conventionalpolyethylene bag. In some embodiments, the mixing step takes betweenabout 30 seconds and 5 minutes. In some embodiments, the mixing stepoutlined above can take place in a stainless steel container.

The mixing step using the high shear mixing apparatus can take place inany conventional high shear mixing apparatus. An example of such a highshear mixing apparatus is sold as Lab High Shear Granulator (KeyInternational, Inc., Englishtown, N.J.). In some embodiments, the mixingis performed for less than about 10 minutes. In some other embodiments,the mixing is performed for less than about 5 minutes.

The capsule filling step outlined above can take place in anyconventional capsule filling system or apparatus. In some embodiments,the capsule filling system is semi-automated, and can handle small batchsizes. An example of such a capsule filling system is sold as In-Cap(Isopak Limited, Lincolnshire, Stamford, United Kingdom). In someembodiments, the capsule filling system is manual. An example of such acapsule filling apparatus is sold as ProFill 100 (Torpac, Inc.,Fairfield, N.J., USA).

In some embodiments, the capsules are hard gelatin capsules, sold asConi-Snap® (Capsugel, Peapack, N.J.). One of skill in the art will beable to select the appropriate capsule size and color. In someembodiments, the capsules have a fill weight of 85 mg, 120 mg, or 150mg.

The weight-sorting step outlined above can take place using anyconventional weight-sorting apparatus or machine. An example of aweight-sorting apparatus or machine is sold as the SADE SP Bench TopTablet and Capsule Weight Sorter (AC Compacting LLC, North Brunswick,N.J., USA).

In some embodiments, the capsules are packaged in bottles, foil pouchesor blister packs. In some other embodiments, the capsules are packagedin heat-induction sealed high-density polyethylene (HDPE) bottles. Inanother embodiment, the capsules are packaged in air-tight sealed foilpouches. In yet another embodiment, the capsules are packaged infoil-foil blister packs. In some other embodiments, the capsules arepackaged with a desiccant.

The physical and chemical stability of the oral pharmaceutical dosageform may be tested in a conventional manner, for example, themeasurement of dissolution, disintegration time, assay for the compoundof formula (I) degradation products, after storage at differenttemperatures for different lengths of time.

In some other embodiments, the invention provides pharmaceuticalcompositions for parenteral use. In yet some other embodiments, theinvention provides liquid pharmaceutical compositions for parenteral ororal use.

In some embodiments, the compound of formula (I) is formulated as alyophilized powder, in a manner analogous to that described in Plamondonet al., WO 02/059131, hereby incorporated by reference in its entirety.In such embodiments, an aqueous mixture comprising a alpha-hydroxycarboxylic acid or a beta-hydroxy carboxylic acid is lyophilized to formthe compound of formula (I).

In some embodiments, the lyophilized powder also comprises freealpha-hydroxy carboxylic acid, or beta hydroxy carboxylic acid.Preferably, the free alpha-hydroxy carboxylic acid or beta hydroxycarboxylic acid compound and the compound of formula (I) are present inthe mixture in a molar ratio ranging from about 0.5:1 to about 100:1,more preferably from about 5:1 to about 100:1. In various embodimentswherein the alpha-hydroxy carboxylic acid or beta-hydroxy carboxylicacid compound is citric acid, the lyophilized powder comprises freecitric acid and the corresponding boronate ester in a molar ratioranging from about 10:1 to about 100:1, from about 20:1 to about 100:1,or from about 40:1 to about 100:1.

In some embodiments, the lyophilized powder comprises citric acid and acompound of formula (I), substantially free of other components.However, the composition can further comprise one or more otherpharmaceutically acceptable excipients, carriers, diluents, fillers,salts, buffers, bulking agents, stabilizers, solubilizers, and othermaterials well known in the art. The preparation of pharmaceuticallyacceptable formulations containing these materials is described in,e.g., Remington: The Science and Practice of Pharmacy, 20th Ed., ed. A.Gennaro, Lippincott Williams & Wilkins, 2000, or latest edition, andStrickley, Pharmaceutical Research, 21(2) 201-230 (2004).

Upon dissolution in aqueous medium, equilibrium is established betweenthe boronate ester compound of formula (I) and the corresponding freeboronic acid compound. In some embodiments, equilibrium is reachedquickly, e.g., within 1-15 minutes, after the addition of aqueousmedium. The relative concentrations of boronate ester, boronic acid, andany intermediate species present at equilibrium is dependent uponparameters such as, e.g., the pH of the solution, temperature, thenature of the alpha-hydroxy carboxylic acid or beta-hydroxy carboxylicacid, and the ratio of the alpha-hydroxy carboxylic acid or beta-hydroxycarboxylic acid to boronate ester compound of formula (I) present in thelyophilized powder.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I), a bulking agent, and a buffer. In some otherembodiments, the pharmaceutical composition comprises the compound offormula (I), a bulking agent, and a buffer in a lyophilized powder.

In some embodiments, the compound of formula (I) is pre-formed. In someother embodiments, the compound of formula (I) is formed in situ, fromthe corresponding boronic acid of formula (VIII). In yet some otherembodiments, the compound (I-1) is pre-formed. In still yet some otherembodiments, the compound (I-15) is formed in situ from compound(VIII-15).

Suitable bulking agents include glycine. In some embodiments, the amountof the bulking agent present is about 1% weight/volume (w/v) to about 5%w/v. In some other embodiments, the amount of the bulking agent presentis about 3% w/v.

Suitable buffers include sodium citrate, citric acid, and mixturesthereof. In some embodiments, the buffer is sodium citrate, and citricacid.

In some embodiments, the buffer is present in a concentration of about45 mM to about 65 mM. In some other embodiments, the buffer is presentin a concentration of about 50 mM to about 60 mM.

In some embodiments, the ratio of the buffer to the compound of formula(I) is from about 50:1 to about 10:1. In some other embodiments, theratio of the buffer to the compound of formula (I) is from about 30:1 toabout 10:1. In yet some other embodiments, the ratio of the buffer tothe compound of formula (I) is about 20:1.

In some embodiments, the pH of the pharmaceutical composition is betweenabout pH 4.7 and pH 6.1. The pH of the pharmaceutical composition can beadjusted using any suitable inorganic acid or organic acid.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I), a bulking agent, and a buffer; wherein:

-   -   the alpha-hydroxy carboxylic acid or beta-hydroxy carboxylic        acid is citric acid;    -   A is 0;    -   R^(a) is isobutyl;    -   R^(a1) is hydrogen, C₁₋₆ aliphatic, —(CH₂)_(m)—CH₂—R^(B), or        —(CH₂)_(m)—CH(R^(5a))—OR^(5b);    -   P is R^(c)—C(O)—;    -   R^(c) is —R^(D);    -   m is 0 or 1;    -   the bulking agent is glycine; and    -   the buffer is sodium citrate, and citric acid.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I), a bulking agent, and a buffer, wherein:

-   -   the compound of formula (I) is represented by compounds (I-1),        (I-15) or (I-18);    -   the bulking agent is glycine; and    -   the buffer is sodium citrate, and citric acid.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I), a bulking agent, and a buffer in a lyophilizedpowder; wherein:

-   -   the alpha-hydroxy carboxylic acid or beta-hydroxy carboxylic        acid is citric acid;    -   A is 0;    -   R^(a) is isobutyl;    -   R^(a1) is hydrogen, C₁₋₆ aliphatic, —(CH₂)_(m)—CH₂—R^(B), or        —(CH₂)_(m)—CH(R^(5a))—OR^(5b);    -   P is R^(c)—C(O)—;    -   R^(c) is —R^(D);    -   m is 0 or 1;    -   the bulking agent is glycine; and    -   the buffer is sodium citrate and citric acid.

In some embodiments, the pharmaceutical composition comprises thecompound of formula (I), a bulking agent, and a buffer in a lyophilizedpowder, wherein:

-   -   the compound of formula (I) is represented by compounds (I-1),        (I-15) or (I-18);    -   the bulking agent is glycine; and    -   the buffer is sodium citrate and citric acid.

In some embodiments, the pharmaceutical composition comprises thecompound (I-1) in a lyophilized powder. In some other embodiments, thepharmaceutical composition comprises the compound (I-1), glycine, sodiumcitrate, and citric acid in a lyophilized powder. In yet some otherembodiments, the pharmaceutical composition comprises the compound(I-15) in a lyophilized powder. In still yet some other embodiments, thepharmaceutical composition comprises the compound (I-15), glycine,sodium citrate, and citric acid in a lyophilized powder.

In some embodiments, the invention provides a unit dose pharmaceuticalcomposition comprising the compound of formula (I-1), a bulking agent,and a buffer in a lyophilized powder. In some embodiments, the unit dosepharmaceutical composition comprises the compound of formula (I-1),glycine, sodium citrate, and citric acid in a lyophilized powder.

In some embodiments, the compound of formula (I-1) is present in theunit dose pharmaceutical composition in an amount equivalent on a molarweight basis of about 1 mg to about 10 mg of the compound of formula(VIII-1). In some embodiments, the compound of formula (I-1) is presentin the unit dose pharmaceutical composition in an amount equivalent on amolar weight basis of about 1 mg to about 5 mg of the compound offormula (VIII-1). In some embodiments, the compound of formula (I-1) ispresent in the unit dose pharmaceutical composition in an amountequivalent on a molar weight basis of about 1.0 mg, about 1.5 mg, about2.0 mg, about 2.5 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, or about5.0 mg of the compound of formula (VIII-1). In some embodiments, thecompound of formula (I-1) is present in the unit dose pharmaceuticalcomposition in an amount equivalent on a molar weight basis of about 3.5mg of the compound of formula (VIII-1).

In some embodiments, the amount of glycine present in the unit dosepharmaceutical composition is about 0.01 g to about 0.50 g. In someembodiments, the amount of glycine present in the unit dosepharmaceutical composition is about 0.03 g to about 0.250 g. In someembodiments, the amount of glycine present in the unit dosepharmaceutical composition is about 0.06 g to about 0.125 g.

In some embodiments, the sodium citrate and citric acid is present inthe unit dose pharmaceutical composition in an amount equivalent toabout 0.005 g to about 0.250 g of citrate ion. In some embodiments, thesodium citrate and citric acid is present in the unit dosepharmaceutical composition in an amount equivalent to about 0.025 g toabout 0.125 g of citrate ion.

In some embodiments, the invention provides a unit dose pharmaceuticalcomposition comprising the compound of formula (I-15), a bulking agent,and a buffer in a lyophilized powder. In some embodiments, the unit dosepharmaceutical composition comprises the compound of formula (I-15),glycine, sodium citrate, and citric acid in a lyophilized powder.

In some embodiments, the compound of formula (I-15) is present in theunit dose pharmaceutical composition in an amount equivalent on a molarweight basis of about 1 mg to about 10 mg of the compound of formula(VIII-15). In some embodiments, the compound of formula (I-15) ispresent in the unit dose pharmaceutical composition in an amountequivalent on a molar weight basis of about 1 mg to about 5 mg of thecompound of formula (VIII-1). In some embodiments, the compound offormula (I-15) is present in the unit dose pharmaceutical composition inan amount equivalent on a molar weight basis of about 1.0 mg, about 1.5mg, about 2.0 mg, about 2.5 mg, about 3.5 mg, about 4.0 mg, about 4.5mg, or about 5.0 mg of the compound of formula (VIII-15). In someembodiments, the compound of formula (I-15) is present in the unit dosepharmaceutical composition in an amount equivalent on a molar weightbasis of about 3.5 mg of the compound of formula (VIII-15).

In some embodiments, the amount of glycine present in the unit dosepharmaceutical composition is about 0.01 g to about 0.50 g. In someembodiments, the amount of glycine present in the unit dosepharmaceutical composition is about 0.03 g to about 0.250 g. In someembodiments, the amount of glycine present in the unit dosepharmaceutical composition is about 0.06 g to about 0.125 g.

In some embodiments, the sodium citrate and citric acid is present inthe unit dose pharmaceutical composition in an amount equivalent toabout 0.005 g to about 0250 g of citrate ion. In some embodiments, thesodium citrate and citric acid is present in the unit dosepharmaceutical composition in an amount equivalent to about 0.025 g toabout 0.125 g of citrate ion.

In another aspect, the invention provides a method for preparing thecompound of formula (I) as a lyophilized powder, the method comprisingthe steps:

-   -   (d-1) combining:        -   i. an aqueous solvent mixture;        -   ii. the compound of formula (I);        -   iii. a bulking agent; and        -   iv. a buffer; to form a mixture; and    -   (d-2) lyophilizing the mixture.

In some embodiments, the compound of formula (I) is formed in situ fromthe corresponding compound of formula (VIII). Thus, the invention alsoprovides a method for preparing the compound of formula (I) as alyophilized powder, the method comprising the steps:

-   -   (e-1) combining:        -   i. an aqueous solvent mixture;        -   ii. the compound of formula (V/I);        -   iii. a bulking agent; and        -   iv. an alpha-hydroxy carboxylic acid, or a salt thereof; or            a beta-hydroxy carboxylic acid, or a salt thereof; or a            combination thereof; to form a mixture; and    -   (e-2) lyophilizing the mixture.

In some embodiments, the aqueous solvent mixture comprises one or moreco-solvents in addition to water. In some embodiments, the co-solvent ismiscible with water. In some other embodiments, the co-solvent is analcohol including, but not limited to, ethanol, tert-butyl alcohol andmixtures thereof. In some other embodiments, the co-solvent istert-butyl alcohol.

In some embodiments, the aqueous solvent mixture comprises about 1% v/vto about 40% v/v alcohol. In some other embodiments, the aqueous solventmixture comprises about 3% v/v to about 10% v/v alcohol. In some otherembodiments, the aqueous solvent mixture comprises about 3% v/v to about6% v/v alcohol. In yet some other embodiments, the solvent mixturecomprises about 3% v/v to about 6% v/v tert-butyl alcohol. In still yetsome other embodiments, the solvent mixture comprises about 5% v/vtert-butyl alcohol.

In some embodiments, a method is provided for preparing the compound(I-1) as a lyophilized powder, the method comprising the steps:

-   -   (f-1) combining:        -   i. water;        -   ii. the compound (I-1);        -   iii. glycine;        -   iv. sodium citrate; and        -   v. citric acid; to form a mixture; and    -   (f-2) lyophilizing the mixture.

In some embodiments, a method is provided for preparing the compound(I-15) as a lyophilized powder, the method comprising the steps:

-   -   (g-1) combining:        -   i. an aqueous solvent mixture comprising water and            tert-butyl alcohol;        -   ii. the compound (VIII-1);        -   iii. glycine;        -   iv. sodium citrate; and        -   v. citric acid; to form a mixture; and    -   (g-2) lyophilizing the mixture.

In some other embodiments, for the method described directly above, theamount of tert-butyl alcohol present in the aqueous solvent mixture isabout 3% v/v to about 6% v/v.

The lyophilization or freeze-drying can be performed using anyconventional lyophilizers or freeze-dryers. In some embodiments, thelyophilization comprises the steps: (i) loading the liquid mixture asprepared above, and freezing; (ii) annealing; (iii) second freeze cycle;(iv) drying under vacuum; and (v) secondary drying. The temperatures andtimes for each step will depend on the lyophilizer or freeze-dryer thatis employed.

In some embodiments, the resulting lyophilized powder has a residualmoisture content of less than about 2%. In some other embodiments, theresulting lyophilized powder has a residual moisture content of lessthan about 1%.

In another aspect, the invention provides a method for the preparationof a pharmaceutical composition of the compound of formula (I) as aliquid pharmaceutical dosage form, said method comprising the step ofreconstituting a lyophilized powder of the compound of formula (I) withan aqueous solvent suitable for pharmaceutical administration. Suitablereconstitution solvents include, but are not limited to, water, saline,phosphate buffered saline (PBS), and mixtures thereof. In someembodiments, the reconstitution solvent is water, water for injection,saline, and mixtures thereof. In some other embodiments, thereconstitution solvent is water for injection. Following reconstitution,the liquid pharmaceutical dosage form can contain concentrations of thecompound of formula (I) as described herein.

In some embodiments, a method is provided for the preparation of apharmaceutical composition of the compound (I-1) as a liquidpharmaceutical dosage form, said method comprising the step ofreconstituting a lyophilized powder of the compound (I-1) as describedherein with an aqueous solvent suitable for pharmaceuticaladministration. In some embodiments, a method is provided for thepreparation of a pharmaceutical composition of the compound (I-1) as aliquid pharmaceutical dosage form, said method comprising the step ofreconstituting a lyophilized powder of the compound (I-1) as describedherein with water for injection, or normal saline. In some embodiments,a method is provided for the preparation of a pharmaceutical compositionof the compound (I-1) as a liquid pharmaceutical dosage form, saidmethod comprising the step of reconstituting a lyophilized powder of thecompound (I-1) as described herein with water for injection.

In some embodiments, a method is provided for the preparation of apharmaceutical composition of the compound (I-15) as a liquidpharmaceutical dosage form, said method comprising the step ofreconstituting a lyophilized powder of the compound (I-15) as describedherein with an aqueous solvent suitable for pharmaceuticaladministration. In some embodiments, a method is provided for thepreparation of a pharmaceutical composition of the compound (I-15) as aliquid pharmaceutical dosage form, said method comprising the step ofreconstituting a lyophilized powder of the compound (I-15) as describedherein with water for injection, or normal saline. In some embodiments,a method is provided for the preparation of a pharmaceutical compositionof the compound (I-15) as a liquid pharmaceutical dosage form, saidmethod comprising the step of reconstituting a lyophilized powder of thecompound (I-15) as described herein with water for injection.

Upon reconstitution in a reconstitution solvent, an equilibrium isestablished between the compound of formula (I) and the correspondingboronic acid of formula (VIII). Typically, equilibrium is reachedquickly within about 10-15 minutes after the addition of thereconstitution solvent. The relative concentrations of the boronateester and boronic acid present at equilibrium is dependent upon the pHof the solution, temperature, and the ratio of the alpha-hydroxy orbeta-hydroxy acid compound to boronic acid compound.

In another aspect, the invention provides a liquid pharmaceuticalcomposition comprising the compound of formula (I), and additionalexcipients described herein. In some embodiments, the liquidpharmaceutical composition is suitable for parenteral use. In some otherembodiments, the liquid pharmaceutical composition is suitable for oraluse.

In such embodiments, the liquid pharmaceutical composition comprises thecompound of formula (I), a buffer, and optionally a tonicity modifier.

In some embodiments, the ratio of the buffer to the compound of formula(I) is from about 50:1 to about 10:1. In some other embodiments, theratio of the buffer to the compound of formula (I) is from about 30:1 toabout 10:1. In yet some other embodiments, the ratio of the buffer tothe compound of formula (I) is about 20:1.

In some embodiments, the buffer is present in a concentration of about45 mM to about 65 mM. In some other embodiments, the buffer is presentin a concentration of about 50 mM to about 60 mM.

Suitable buffers include sodium citrate, citric acid, and mixturesthereof. In some embodiments, the buffer is sodium citrate, and citricacid.

Suitable tonicity modifiers include, but are not limited to, amino acidssuch as arginine, histidine, and glycine; salts such as sodium chloride,potassium chloride, sodium citrate, propylene glycol; and mixturesthereof. In some embodiments, the tonicity modifier is propylene glycol.In some other embodiments, the tonicity modifier is sodium chloride.

Upon dissolution in a aqueous solvent mixture, an equilibrium isestablished between the compound of formula (I) and the correspondingboronic acid of formula (VIII). Thus, either a compound of formula (I)or a compound of formula (VIII) may be used in the preparation of theliquid pharmaceutical composition. Typically, equilibrium is reachedquickly within about 10-15 minutes after the addition of the aqueoussolvent mixture. The relative concentrations of the boronate ester andboronic acid present at equilibrium is dependent upon the pH of thesolution, temperature, and the ratio of the alpha-hydroxy orbeta-hydroxy acid compound to boronic acid compound. In someembodiments, the excess alpha-hydroxy or beta-hydroxy acid may act as astabilizer, which pushes the equilibrium towards the boronate ester. Insome embodiments, the tonicity modifier may also act as a stabilizer.

In some embodiments, the liquid pharmaceutical composition optionallyfurther comprises a preservative.

In some embodiments, the liquid pharmaceutical composition comprises thecompound of formula (I), a buffer, and optionally a tonicity modifier;wherein:

-   -   the alpha-hydroxy carboxylic acid or beta-hydroxy carboxylic        acid is citric acid;    -   A is 0;    -   R^(a) is isobutyl;    -   R^(a1) is hydrogen, C₁₋₆ aliphatic, —(CH₂)_(m)—CH₂—R^(B), or        —(CH₂)_(m)—CH(R^(5a))—OR^(5b);    -   P is R^(c)—C(O)—;    -   R^(c) is —R^(D);    -   m is 0 or 1;    -   the buffer is sodium citrate and citric acid; and    -   the tonicity modifier, when present, is sodium chloride.

In some embodiments, the liquid pharmaceutical composition comprises thecompound of formula (I), a buffer, and optionally a tonicity modifier;wherein:

-   -   the compound of formula (I) is represented by compounds (I-1),        (I-15) or (I-18);    -   the buffer is sodium citrate and citric acid; and    -   the tonicity modifier, when present, is sodium chloride.

In some embodiments, wherein the alpha-hydroxy carboxylic acid orbeta-hydroxy carboxylic acid is citric acid, the liquid pharmaceuticalcomposition of the compound of formula (I) comprises the compound offormula (I), water, citric acid, sodium citrate, and sodium chloride. Insome other embodiments, wherein the alpha-hydroxy carboxylic acid orbeta-hydroxy carboxylic acid is citric acid, the liquid pharmaceuticalcomposition comprises the compound of formula (I), water, citric acid,and propylene glycol. In yet some other embodiments, the liquidpharmaceutical composition comprises the compound of formula (I),wherein the compound of formula (I) is the compound (I-1), water, citricacid, sodium citrate and sodium chloride.

In such embodiments, wherein the alpha-hydroxy carboxylic acid orbeta-hydroxy carboxylic acid is citric acid, the liquid pharmaceuticaldosage form of the compound of formula (I) has a pH of between about pH3 and about pH 7. In certain such embodiments, the pH is between aboutpH 4.9 and about pH 6.7. In other certain such embodiments, the pH isbetween about pH 5.5 and about pH 6.5.

In some embodiments, wherein the alpha-hydroxy carboxylic acid orbeta-hydroxy carboxylic acid is citric acid, the liquid pharmaceuticalcomposition of the compound of formula (I) is prepared in situ from astock vehicle solution and the compound of formula (VIII). In someembodiments, the stock vehicle solution comprises water, citric acid,sodium citrate and propylene glycol. In such embodiments, the resultingsolution can be further diluted with stock vehicle solution or with asodium chloride solution to generate liquid pharmaceutical compositionsof the compound of formula (I) of desired concentrations.

In another aspect the invention provides a unit dose liquidpharmaceutical composition, comprising the compound of formula (I), abuffer, and optionally a tonicity modifier. In some embodiments, theunit dose liquid pharmaceutical composition comprises the compound offormula (I), a buffer, and optionally a tonicity modifier, wherein thecompound of formula (I) is the compound (I-1). In some embodiments, thecompound of formula (I) is present in the unit dose liquidpharmaceutical composition at a concentration of about 0.5 mg/ml toabout 3 mg/ml of the compound of formula (VIII). In some otherembodiments, the compound of formula (I) is present in the unit doseliquid pharmaceutical composition at a concentration of about 1 mg/ml ofthe compound of formula (VIII). In some other embodiments, wherein thecompound of formula (I) is compound (I-1), the compound (I-1) in theunit dose liquid pharmaceutical composition is present at aconcentration of about 0.5 mg/ml to about 3 mg/ml of the compound offormula (VIII-1). In yet some other embodiments, wherein the compound offormula (I) is compound (I-1), the compound (I-1) in the unit doseliquid pharmaceutical composition is present at a concentration of about1 mg/ml of the compound of formula (VIII-1). In still yet some otherembodiments, wherein the compound of formula (I) is the compound (I-15),the compound (I-15) in the unit dose liquid pharmaceutical compositionis present at a concentration of about 1 mg/ml of the compound offormula (VIII-15).

In some embodiments in the unit dose liquid pharmaceutical composition,the sodium citrate and citric acid are present in an amount equivalentto about 0.005 g to about 0.250 g of citrate ion. In some embodiments inthe unit dose liquid pharmaceutical composition, the sodium citrate andcitric acid are present in an amount equivalent to about 0.025 g toabout 0.125 g of citrate ion.

In some embodiments in the unit dose liquid pharmaceutical composition,the sodium chloride is present in an amount of about 0.0045 g to about0.09 g. In some embodiments in the unit dose liquid pharmaceuticalcomposition, the sodium chloride is present in an amount of about 0.01 gto about 0.04 g.

In some embodiments in the unit dose liquid pharmaceutical composition,the pharmaceutical composition is stored frozen until use.

In another aspect the invention provides a method for preparing thecompound of formula (I), as a unit dose liquid pharmaceuticalcomposition; the method comprising the steps:

-   -   (h-1) dissolving the buffer in an aqueous solvent;    -   (h-2) dissolving the compound of formula (I), or a crystalline        form thereof, in the mixture obtained in step (h-1);    -   (h-3) dissolving the tonicity modifier in the mixture obtained        in step (h-2);    -   (h-4) adding further aqueous solvent to the required batch        volume; and    -   (h-5) filling vials with an amount of the mixture obtained in        step (h-4).

In some embodiments, the vials are capped after step (h-5). In someother embodiments, nitrogen is bubbled through the mixture prior to step(h-5). In yet some other embodiments, after step (h-5), the liquid inthe vials can be overlayed with nitrogen prior to capping.

In some embodiments, the compound of formula (I) is formed in situ fromthe compound of formula (VIII). In such embodiments, in step (h-2), thecompound of formula (VIII), or a crystalline form thereof is added tothe mixture. In some embodiments, the alpha-hydroxy acid or beta-hydroxyacid is added in step (h-2). In some other embodiments, thealpha-hydroxy acid or beta-hydroxy acid is present in step (h-1) as thebuffer.

The pharmaceutical compositions of the invention preferably areformulated for administration to a patient having, or at risk ofdeveloping or experiencing a recurrence of, a proteasome-mediateddisorder. The term “patient”, as used herein, means an animal,preferably a mammal, more preferably a human. Preferred pharmaceuticalcompositions of the invention are those formulated for oral,intravenous, or subcutaneous administration. However, any of the abovedosage forms containing a therapeutically effective amount of a compoundof the invention are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention. In someembodiments, the pharmaceutical composition of the invention may furthercomprise another therapeutic agent. In some embodiments, such othertherapeutic agent is one that is normally administered to patients withthe disease or condition being treated.

By “therapeutically effective amount” is meant an amount sufficient tocause a detectable decrease in proteasome activity or the severity of aproteasome-mediated disorder. The amount of proteasome inhibitor neededwill depend on the effectiveness of the inhibitor for the given celltype and the length of time required to treat the disorder. It shouldalso be understood that a specific dosage and treatment regimen for anyparticular patient will depend upon a variety of factors, including theactivity of the specific compound employed, the age, body weight,general health, sex, and diet of the patient, time of administration,rate of excretion, drug combinations, the judgment of the treatingphysician, and the severity of the particular disease being treated. Theamount of additional therapeutic agent present in a composition of thisinvention typically will be no more than the amount that would normallybe administered in a composition comprising that therapeutic agent asthe only active agent. Preferably, the amount of additional therapeuticagent will range from about 50% to about 100% of the amount normallypresent in a composition comprising that agent as the onlytherapeutically active agent.

In another aspect, the invention provides a method for treating apatient having, or at risk of developing or experiencing a recurrenceof, a proteasome-mediated disorder. As used herein, the term“proteasome-mediated disorder” includes any disorder, disease orcondition which is caused or characterized by an increase in proteasomeexpression or activity, or which requires proteasome activity. The term“proteasome-mediated disorder” also includes any disorder, disease orcondition in which inhibition of proteasome activity is beneficial.

For example, compounds and pharmaceutical compositions of the inventionare useful in treatment of disorders mediated via proteins (e.g., NFκB,p27^(Kip), p21^(WAF/CIP1), p53) which are regulated by proteasomeactivity. Relevant disorders include inflammatory disorders (e.g.,rheumatoid arthritis, inflammatory bowel disease, asthma, chronicobstructive pulmonary disease (COPD), osteoarthritis, dermatosis (e.g.,atopic dermatitis, psoriasis)), vascular proliferative disorders (e.g.,atherosclerosis, restenosis), proliferative ocular disorders (e.g.,diabetic retinopathy), benign proliferative disorders (e.g.,hemangiomas), autoimmune diseases (e.g., multiple sclerosis, tissue andorgan rejection), as well as inflammation associated with infection(e.g., immune responses), neurodegenerative disorders (e.g., Alzheimer'sdisease, Parkinson's disease, motor neurone disease, neuropathic pain,triplet repeat disorders, astrocytoma, and neurodegeneration as resultof alcoholic liver disease), ischemic injury (e.g., stroke), andcachexia (e.g., accelerated muscle protein breakdown that accompaniesvarious physiological and pathological states, (e.g., nerve injury,fasting, fever, acidosis, HIV infection, cancer affliction, and certainendocrinopathies)).

The compounds and pharmaceutical compositions of the invention areparticularly useful for the treatment of cancer. As used herein, theterm “cancer” refers to a cellular disorder characterized byuncontrolled or disregulated cell proliferation, decreased cellulardifferentiation, inappropriate ability to invade surrounding tissue,and/or ability to establish new growth at ectopic sites. The term“cancer” includes, but is not limited to, solid tumors and bloodbornetumors. The term “cancer” encompasses diseases of skin, tissues, organs,bone, cartilage, blood, and vessels. The term “cancer” furtherencompasses primary and metastatic cancers.

Non-limiting examples of solid tumors that can be treated with thedisclosed proteasome inhibitors or pharmaceutical compositions includepancreatic cancer; bladder cancer; colorectal cancer; breast cancer,including metastatic breast cancer; prostate cancer, includingandrogen-dependent and androgen-independent prostate cancer; renalcancer, including, e.g., metastatic renal cell carcinoma; hepatocellularcancer; lung cancer, including, e.g., non-small cell lung cancer(NSCLC), bronchioloalveolar carcinoma (BAC), and adenocarcinoma of thelung; ovarian cancer, including, e.g., progressive epithelial or primaryperitoneal cancer; cervical cancer, gastric cancer; esophageal cancer;head and neck cancer, including, e.g., squamous cell carcinoma of thehead and neck; melanoma; neuroendocrine cancer, including metastaticneuroendocrine tumors; brain tumors, including, e.g., glioma, anaplasticoligodendroglioma, adult glioblastoma multiforme, and adult anaplasticastrocytoma; bone cancer; and soft tissue sarcoma.

Non-limiting examples of hematologic malignancies that can be treatedwith the disclosed proteasome inhibitors or pharmaceutical compositionsinclude acute myeloid leukemia (AML); chronic myelogenous leukemia(CML), including accelerated CML and CML blast phase (CML-BP); acutelymphoblastic leukemia (ALL); chronic lymphocytic leukemia (CLL);Hodgkin's disease (HD); non-Hodgkin's lymphoma (NHL), includingfollicular lymphoma and mantle cell lymphoma; B-cell lymphoma; T-celllymphoma; multiple myeloma (MM); Waldenstrom's macroglobulinemia;myelodysplastic syndromes (MDS), including refractory anemia (RA),refractory anemia with ringed siderblasts (RARS), (refractory anemiawith excess blasts (RAEB), and RAEB in transformation (RAEB-T); andmyeloproliferative syndromes.

In some embodiments, the compound or pharmaceutical compositions of theinvention are used to treat a patient having or at risk of developing orexperiencing a recurrence in a cancer selected from the group consistingof multiple myeloma and mantle cell lymphoma.

In some embodiments, the proteasome inhibitor or pharmaceuticalcompositions of the invention is administered in conjunction withanother therapeutic agent. The other therapeutic agent may also inhibitthe proteasome, or may operate by a different mechanism. In someembodiments, the other therapeutic agent is one that is normallyadministered to patients with the disease or condition being treated.The proteasome inhibitor of the invention may be administered with theother therapeutic agent in a single dosage form or as a separate dosageform. When administered as a separate dosage form, the other therapeuticagent may be administered prior to, at the same time as, or followingadministration of the proteasome inhibitor of the invention.

In some embodiments, a proteasome inhibitor of formula (I), orpharmaceutical composition of the compound of formula (I) isadministered in conjunction with an anticancer agent. As used herein,the term “anticancer agent” refers to any agent that is administered toa subject with cancer for purposes of treating the cancer.

Non-limiting examples of DNA damaging chemotherapeutic agents includetopoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecinand analogs or metabolites thereof, and doxorubicin); topoisomerase IIinhibitors (e.g., etoposide, teniposide, and daunorubicin); alkylatingagents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide,carmustine, lomustine, semustine, streptozocin, decarbazine,methotrexate, mitomycin C, and cyclophosphamide); DNA intercalators(e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators andfree radical generators such as bleomycin; and nucleoside mimetics(e.g., 5-fluorouracil, capecitibine, gemcitabine, fludarabine,cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include:paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, andrelated analogs; thalidomide, lenalidomide, and related analogs (e.g.,CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinibmesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κBinhibitors, including inhibitors of IκB kinase; antibodies which bind toproteins overexpressed in cancers and thereby downregulate cellreplication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab);and other inhibitors of proteins or enzymes known to be upregulated,over-expressed or activated in cancers, the inhibition of whichdownregulates cell replication.

In order that this invention be more fully understood, the followingpreparative and testing examples are set forth. These examplesillustrate how to make or test specific compounds, and are not to beconstrued as limiting the scope of the invention in any way.

EXAMPLES Abbreviations

DCM methylene chlorideDIPEA N,N′-diisopropylethyl amineDMF N,N′-dimethylformamideEDCI N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlorideEtOAc ethyl acetateh hoursHPLC high performance liquid chromatographyMIBK methyl isobutyl ketonePES polyethersulfoneTBTU O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborateTFA trifluoroacetic acidTHF tetrahydrofuranHOBt 1-hydroxybenztriazoleLCMS liquid chromatography mass spectrummin minutes

General Methods

¹H NMR:

The spectra are acquired at ambient temperature on a JOEL ECX-400 NMRspectrometer operating at 400 MHz for ¹H NMR. The resulting FID's aretransferred to a PC and processed using NUTS NMR processing softwarefrom Acorn NMR Inc. Chemical shifts are referenced to DMSO solvent, 2.50ppm. A solvent blank is prepared by adding ˜0.75 mL of DMSO-d6 to theNMR tube. After a ¹H spectrum is acquired on the solvent blank, thesample is added and completely dissolved.

Mass Spectrometry:

Mass spectrometry studies are run on a Thermo-Finnigan LCQ Deca-XP iontrap mass spectrometer. The electrospray ion source was used in bothpositive and negative modes with a high voltage of 5 kv, sheath gas flowrate of 35 arb, capillary temperature of 275° C., capillary voltage of 9V and tube lens offset of 35 V. An analyte was dissolved in acetonitrileto generate a 0.5 mg/ml solution. An Agilent 1100 HPLC system was usedfor LC-Mass spectrometry flow analysis. The pump flow rate was 1.0ml/minute. 10 μl of each sample solution was injected from theautosampler into a T-joint. About 2% of the solution from the T-jointwas infused into the mass spectrometer.

X-Ray Powder Diffractometry (XRPD):

X-ray powder diffraction patterns are acquired on either:

i) a Bruker AXS D8Advance diffractometer. The data is collected over anangular range of 2.9° to 29.6° 2θ in continuous scan mode using a stepsize of 0.05° 2θ and a step time of 2 seconds. The sample is run underambient conditions and prepared as a flat plate specimen using powder asreceived without grinding; orii) a PANalytical X'Pert Pro diffractometer. Each specimen is analyzedusing Cu radiation produced using an Optix long fine-focus source. Anelliptically graded multilayer mirror is used to focus the Cu K α X-raysof the source through the specimen and onto the detector. The specimenis sandwiched between 3-micron thick films, analyzed in transmissiongeometry, and rotated to optimize orientation statistics. A beam-stop isused to minimize the background generated by air scattering. Helium andthe anti-scatter extension are not used. Soller slits are used for theincident and diffracted beams to minimize axial divergence. Thediffraction patterns are collected using a scanning position-minimizeaxial divergence. The diffraction patterns are collected using ascanning position-sensitive detector (X'Celerator) located 240 mm fromthe specimen. Prior to the analyses, a silicon specimen (NIST standardreference material 640c) is analyzed to verify the position of thesilicon 111 peak.

Differential Scanning Calorimetry (DSC):

Differential scanning calorimetry (DSC) data are collected on either:

i) a TA Instruments Q100 differential scanning calorimeter equipped witha 50 position auto-sampler. The energy and temperature calibrationstandard is indium. Samples are heated at a rate of 10° C. per minutebetween 25° C. and 300° C. A nitrogen purge flowing at 50 mL per minuteis maintained over the sample during a scan. Between 1 mg and 3 mg ofsample is analyzed. All samples are crimped in a hermetically sealedaluminum pan with a pinhole to alleviate the pressure accumulated fromthe solvent vapor; orii) a TA Instruments differential scanning calorimeter 2920. The sampleis placed into an aluminum DSC pan and the weight is accuratelyrecorded. The open pan is covered with a lid and then crimped. Thesample cell is equilibrated at 25° C., and heated under a nitrogen purgeat a rate of 10° C./min. Indium metal was used as the calibrationstandard.

Thermal Gravimetric Analysis (TGA):

Thermal gravimetric analysis (TGA) data are collected on a TAInstruments Q500 thermal gravimetric analyzer, calibrated withNickel/Alumel and running at a scan rate of 10° C. per minute. Anitrogen purge flowing at 60 mL per minute is maintained over the sampleduring measurements. Typically 5 mg to 15 mg of sample is loaded onto apre-tared platinum crucible.

Example 1: Synthesis of4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicAcid (I-1)

Step 1: 2,5-[(dichlorobenzoyl)amino]acetic Acid

To a mixture of NaOH (12 g, 300 mmol) and glycine (18 g, 239 mmol) inwater (120 mL) was added dropwise over 45 min a solution of2,5-dichlorobenzoyl chloride (10 g, 48 mmol) in THF (15 mL) keeping theinternal temperature below about 25° C. After 1 h, the mixture wasacidified with 2.0 M HCl (125 mL) keeping the internal temperature belowabout 5° C. The resulting precipitate was collected by vacuumfiltration. The crude product was recrystallized from water to give2,5-[(dichlorobenzoyl)amino]acetic acid as a white, crystalline solid(6.1 g, 52%). mp 173.3° C. ¹H NMR (300 MHz, DMSO-d₆, δ): 12.72 (bs, 1H),8.89 (t, J=6.0 Hz, 1H), 7.54 (m, 2H), 7.48 (m, 1H), 3.93 (d, J=6.0 Hz).¹³C NMR (75 MHz, DMSO-d₆, δ): 41.6, 129.3, 129.6, 131.4, 132.2, 138.2,171.4, 165.9. MS (m/z): [M+H] calculated for C₉H₈Cl₂NO₃, 248.0. found,248.0; [M+Na] calculated for C₉H₇Cl₂NNaO₃, 270.0. found 270.2.

2,5-[(dichlorobenzoyl)amino]acetic acid was also be prepared via thefollowing procedure: To a mixture of glycine (21.5 g, 286 mmol) in water(437 mL), was added 2.0 M NaOH (130 mL) and the resulting solution wascooled to 0° C. A solution of 2,5-dichlorobenzoyl chloride (50.0 g, 239mmol) in THF (75 mL) was added dropwise at such a rate that the internaltemperature was maintained at 0±1° C. During the addition, the pH wascontrolled at 11.0±0.2 using a pH controller titrated with 2.0 M NaOH.After complete addition, the mixture was stirred at 0±1° C. for anadditional 2 h. The mixture was then acidified with 2.0 M HCl (176 mL)to a final pH of 2.5. The resulting precipitate was collected byfiltration, washed with cold water (125 mL), and dried at 45° C. in avacuum oven to afford 2,5-[(dichlorobenzoyl)amino]acetic acid as a whitesolid (57.6 g, 97.3%).

Step 2:2,5-dichloro-N-[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanol-1,3,2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]benzamide

To a solution of 2,5-[(dichlorobenzoyl)amino]acetic acid (6.10 g, 24.6mmol) and TBTU (8.34 g, 26.0 mmol) in DMF (40 mL) with an internaltemperature below about 5° C. was added(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butan-1-amine.TFA(9.35 g, 24.7 mmol). DIPEA (13 mL, 75 mmol) was then added dropwise over2 h keeping the internal temperature below about 5° C. After 40 min, themixture was diluted with EtOAc (90 mL), washed with 5% NaCl (150 mL),twice with 10% NaCl (2×40 mL), once with 2% K₂CO₃ (1×40 mL), once with1% H₃PO₄ (1×40 mL), and once with 10% NaCl (1×40 mL). The resultingorganic layer was concentrated to a thick oil, diluted with heptane (40mL) and evaporated to yield2,5-dichloro-N-[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]benzamideas a white solid which was used in the next step without purification.

Step 3:N,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)

To a solution of2,5-dichloro-N-[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]benzamide(12.2 g, 24.6 mmol) in methanol/hexane (1:1) (250 mL) were added 1N HCl(30 mL, 30 mmol) and (2-methylpropyl)boronic acid (6.5 g, 64 mmol). Thereaction mixture was allowed to stir overnight. The phases wereseparated and the methanol layer was washed twice with additionalheptane (2×55 mL). The resulting organic layer was concentrated to about10 mL and partitioned between 2.0M NaOH (30 mL) and DCM (25 mL). The DCMlayer was washed once with additional 2.0M NaOH (5 mL). The basicaqueous layers were then combined, washed twice with DCM (2×25 mL) andacidified with 1M HCl (60 mL). The resulting mixture was diluted withDCM (40 mL), the layers were separated, and the resulting aqueous layerwas washed three times with DCM (3×10 mL). The combined DCM extractswere dried over MgSO4 (25 g) and evaporated to a thick oil. The productwas precipitated with heptane (50 mL) and collected by filtration toyieldN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)as a white solid (6.6 g, 74%). ¹H NMR (300 MHz, DMSO-d₆, δ): 8.93 (t,J=6.0 Hz, 1H), 8.68 (bs, 1H), 7.63 (m, 1H), 7.52 (m, 2H), 4.00 (d, J=6.0Hz, 2H), 2.62 (m, 1H), 1.59 (m, 1H), 1.33 (m, 1H), 1.24 (m, 1H), 0.81(d, J=5.9 Hz, 6H). ¹³C NMR (125 MHz, DMSO-d₆, δ): 232, 25.8, 40.1, 40.7,43.0, 129.0, 130.0, 131.0, 137.5, 165.0, 172.5. MS (m/z) in CH₃CN: [M+H]calculated for C₄₂H₅₂B₃Cl₆N₆O₉, 1027.2. found, 1027.3; [M+Na] calculatedfor C₄₂H₅₁B₃Cl₆N₆NaO₉, 1049.2. found 1049.5.

Step 4:4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicAcid (I-1)

Form 1:

To a solution of citric acid (2.75 g, 14.3 mmol) in EtOAc (85 mL) withan internal temperature of about 74° C. was addedN,N′,N″-{boroxin-2,4,6-tris[[(R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(5.00 g, 4.87 mmol) as a solid. The solution was cooled uncontrolleduntil the internal temperature was about 25° C. and the mixture wasstirred overnight. The resulting precipitate was collected by filtrationto yield2,2′-{2-[(1R)-1-({[(2,5-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]-5-oxo-1,3,2-dioxaborolane-4,4-diyl}diaceticacid Form 1 as a crystalline solid (6.65 g, 88%). ¹H NMR (500 MHz,DMSO-d₆, δ 110° C.): 10.08 (s, 1H), 8.69 (s, 1H), 7.61 (s, 1H), 7.52 (d,J=1.3 Hz, 2H), 4.26 (d, J=5.5 Hz, 2H), 2.70 (q, J=14.5 Hz, 4H), 2.70(bs, 1H), 1.72 (sept, J=6.5 Hz, 1H), 1.42 (ddd, J=5.2 Hz, J=8.6 Hz,J=13.9 Hz, 1H), 1.28 (ddd, J=5.3, J=9.4 Hz, J=14.3 Hz, 1H), 0.91 (dd,J=3.3 Hz, J=6.6 Hz, 6H). MS (m/z) in CH₃CN: [M+Na] calculated forC₂₀H₂₃BCl₂N₂NaO₉, 539.1. found, 539.1.

The XRPD data for I-1 Form 1 is shown in FIG. 1 and in Table 1.

TABLE 1 XRPD Data I-1 Form 1 Angle 2θ° Intensity % 6.441 100 8.304 29.510.35 19 11.619 5.1 12.695 13.6 15.077 28.2 16.352 28.7 17.504 16.318.231 6 19.086 21.4 20.405 11.7 21.231 7.6 21.916 7.6 25.371 15.227.588 6.2

The Differential Scanning Calorimetry (DSC) data for I-1 Form 1 is shownin FIG. 2. The profile is characterized by an endothermic transitionwith an onset temperature of 191.8° C. with a melt of 198.8° C. A secondendothermic transition corresponding to decomposition has an onsettemperature of 225° C. These temperatures have an error of ±5° C.

The Thermal Gravimetric Analysis (TGA) data for I-1 Form 1 is shown inFIG. 2. The profile graphs the percent loss of weight of the sample as afunction of temperature, the temperature rate change being about 10°C./min. The weight loss represents a loss of about 0.72% of the weightof the sample as the temperature is changed from 50° C. to 200° C. Thesetemperatures have an error of ±5° C.

Form 2:

To a solution of citric acid (10.1 g, 52.6 mmol) in EtOAc (300 mL) withan internal temperature of about 74° C. was added a solution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(20.0 g, 19.5 mmol) in EtOAc (60 mL). The solution was cooled slowly(about 0.33° C./min) until the internal temperature was about 60° C. andthe mixture was stirred for 3 h. The resulting slurry was cooled slowly(rate of about 0.12° C./min) until the internal temperature was about25° C. and the mixture was stirred overnight. The resulting precipitatewas collected by filtration to yield4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid Form 2 as a crystalline solid (26.7 g, 98%). ¹H NMR (500 MHz,DMSO-d₆, δ 110° C.): 10.08 (s, 1H), 8.69 (s, 1H), 7.61 (s, 1H), 7.52 (d,J=1.3 Hz, 2H), 4.26 (d, J=5.5 Hz, 2H), 2.70 (q, J=14.5 Hz, 4H), 2.70(bs, 1H), 1.72 (sept, J=6.5 Hz, 1H), 1.42 (ddd, J=5.2 Hz, J=8.6 Hz,J=13.9 Hz, 1H), 1.28 (ddd, J=5.3, J=9.4 Hz, J=14.3 Hz, 1H), 0.91 (dd,J=3.3 Hz, J=6.6 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆, δ 100° C.): 21.65,23.34, 25.09, 38.39, 38.98, 42.07, 76.25, 128.97, 129.14, 130.94,131.48, 131.73, 137.05, 165.44, 170.23, 175.74, 177.43. MS (m/z) inCH₃CN: [M+Na] calculated for C₂₀H₂₃BCl₂N₂NaO₉, 539.1. found, 539.1.

4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid Form 2 was also prepared by adding a solution of citric acid (21 g,0.11 mmol) in THF (80 mL) to a solution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(40 g, 0.11 mmol) in THF (80 mL) at 60° C. The solution was then seededwith Form 2 crystals (400 mg). After stirring for 30 min at 60° C.,EtOAc (400 mL) was added over a period of 9 h. After complete additionof the EtOAc, the temperature was lowered to 20° C. over 5 h. Theresulting suspension was filtered to collect4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid Form 2 as a crystalline solid (40 g, 70%).

4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid Form 2 was also prepared in the same general manner using theconditions described in Table 2.

TABLE 2 Additional conditions for preparation of I-1 Form 2 Initial SeedIsolated Yield Solvent Temperature Temperature I-1 Form 2 acetonitrile80° C. No seeding 77% MIBK 80° C. No seeding 80% 2-methyltetrahydrofuran80° C. 60° C. 72%

4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid Form 2 was also prepared by dissolving in acetone followed byaddition of EtOAc as an antisolvent.

The XRPD data for I-1 Form 2 is shown in FIG. 3 and in Table 3.

TABLE 3 XRPD Data I-1 Form 2 Angle 2-θ ° Intensity % 5.817 100 7.61493.4 11.575 71.1 11.896 67.1 12.571 24.3 14.43 32.2 16.689 65.8 17.36217.8 18.232 53.9 19.596 77.6 19.959 63.8 20.376 36.2 20.998 32.2 21.540.1 21.764 43.4 22.407 77.6 23.12 33.6 23.901 26.3 24.402 20.4 24.88219.7 25.764 19.1 26.464 39.5 27.347 21.7 27.65 17.1 27.979 16.4 29.4120.4

The Differential Scanning Calorimetry (DSC) data for I-1 Form 2 is shownin FIG. 4. The profile is characterized by an endothermic transitionwith an onset temperature of 206.5° C. with a melt of 219.9° C. A secondendothermic transition corresponding to decomposition has an onsettemperature of 225° C. These temperatures have an error of ±5° C.

The Thermal Gravimetric Analysis (TGA) data for I-1 Form 2 is shown inFIG. 4. The profile graphs the percent loss of weight of the sample as afunction of temperature, the temperature rate change being about 10°C./min. The weight loss represents a loss of about 1.1% of the weight ofthe sample as the temperature is changed from 50° C. to 200° C. Thesetemperatures have an error of ±5° C.

Example 1A: Alternate Synthesis of4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicAcid (I-1) Form 2

A 50-L glass reactor equipped with mechanical stirrer, dropping funnel,temperature indicator, and heating/cooling control unit (under nitrogen)was charged with 1.2 micron filtered EtOAc (18.9 kg) and anhydrouscitric acid (0.561 kg, 2.9 mol). The mixture was heated to 71° C. and asolution resulted.N,N′,N″-{(boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(1.109 kg, 3.1 mol) dissolved in EtOAc (4.0 kg) was clarified using anin-line filter (1.2 micron), and the solution was added to the reactionmixture under stirring (193 rpm) over a period of 20 min whilemaintaining a temperature of 73° C. to 75° C. The stirring was reducedto 96 rpm and the mixture was cooled as follows: (1) The mixture waskept at 73° C.-75° C. for 25 min; (2) The mixture was stepwise cooled to40° C. at the rate of approximately 5° C./30 min; (3) The mixture wasallowed to cool uncontrolled overnight to ambient temperature withstirring. The product was then isolated by filtration, washed on thefilter with 1.2 micron filtered EtOAc (2×1.2 kg), and dried under vacuumat 40-41° C. overnight (22 hours) to give 1.458 kg (92%) of the titlecompound. ¹H NMR (400 MHz, DMSO-d₆, δ): 12.13 (s, 2H), 10.69 (s, 1H),9.11 (t, J=5.6 Hz, 1H), 7.66 (t, J=1.2 Hz, 1H), 7.56 (d, J=1.2 Hz, 2H),4.27 (bs, 2H), 2.9-2.55 (m, 5H), 1.67 (bs, 1H), 1.4-1.15 (bs, 2H), 0.86(d, J=6.4 Hz, 6H).

The XRPD data for the compound (I-1) Form 2 is shown in FIG. 7 and inTable 6.

TABLE 6 Angle 2-θ ° Intensity % 5.69 100 7.64 66 9.66 4 11.22 23 11.4251 11.79 37 12.41 15 14.23 15 15.60 6 16.53 32 17.15 4 18.07 31 19.39 5519.79 41 20.24 21 20.79 15 21.36 20 21.61 22 22.23 63 22.55 14 22.97 2023.22 7 23.67 10 23.90 7 24.19 10 24.74 7 24.97 3 25.64 8 26.31 24 26.6410 27.21 7 27.40 7 27.88 5 28.25 4 29.27 11 29.72 10

The Differential Scanning Calorimetry (DSC) data for the compound (I-1)Form 2 is shown in FIG. 8. The profile is characterized by twoendothermic transitions; the first with a melt at about 231.3° C., andthe second with a melt at about 239.9° C. These temperatures have anerror of ±5° C.

Example 2: Synthesis of2,5-dichloro-N-(2-{[(1R)-3-methyl-1-(4-oxo-1,3,2-dioxaborolan-2-yl)butyl]amino}-2-oxoethyl)benzamide(I-2)

To a solution of glycolic acid (0.041 g, 0.54 mmol) in EtOAc (2.0 mL)with an internal temperature of about 60° C. was added a solution ofN,N′,N″-{(boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(0.199 g, 0.19 mmol) in EtOAc (1.0 mL). The solution was cooleduncontrolled until the internal temperature was about 25° C. and thesolvent was removed by evaporation to yield2,5-dichloro-N-(2-{[(1R)-3-methyl-1-(4-oxo-1,3,2-dioxaborolan-2-yl)butyl]amino}-2-oxoethyl)benzamideas a white solid (0.215 g, 95%). MS (m/z) in CH₃CN: [M+Et₃N+H]calculated for C₂₂H₃₅BCl₂N₃O₅, 502.2. found, 502.0. MS (m/z) in CH₃CN:[M−H] calculated for C₁₆H₁₈BCl₂N₂O₅, 399.1. found, 399.0.

Example 3: Synthesis of{(4S)-2-[(1R)-1-({[(2,5-dichlorobenzoyl)amino]-actyl}amino)-3-methylbutyl]-5-oxo-1,3,2-dioxaborolan-4-yl}aceticAcid (I-3)

To a solution of L-malic acid (0.0958 g, 0.714 mmol) in EtOAc (2.0 mL)with an internal temperature of about 60° C. was added a solution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(0.239 g, 0.233 mmol) in EtOAc (1.0 mL). The solution was cooleduncontrolled until the internal temperature was about 25° C. and thesolvent was removed by evaporation to yield{(4S)-2-[(1R)-1-({[(2,5-dichlorobenzoyl)amino]-acetyl}amino)-3-methylbutyl]-5-oxo-1,3,2-dioxaborolan-4-yl}aceticacid as a white solid (0.307 g, 96%). MS (m/z) in CH₃CN: [M+Et₃N+H]calculated for C₂₄H₃₇BCl₂N₃O₇, 560.1. found, 560.1. MS (m/z) in CH₃CN:[M−H] calculated for C₁₈H₂₀BCl₂N₂O₇, 457.1. found, 457.1.

Example 4: Synthesis of2·5-dichloro-N-[2-({(1R)-1-[(4S)-4-cyclohexyl-5-oxo-1,3,2-dioxaborolan-2-yl]-3-methylbutyl}amino)-2-oxoethyl]benzamide(I-4)

To a solution of (S)-hexahydromandelic acid (0.0881 g, 0.557 mmol) inEtOAc (2.0 mL) with an internal temperature of about 60° C. was added asolution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(0.200 g, 0.195 mmol) in EtOAc (1.0 mL). The solution was cooleduncontrolled until the internal temperature was about 25° C. and thesolvent was removed by evaporation to yield2,5-dichloro-N-[2-({(R)-1-[(4S)-4-cyclohexyl-5-oxo-1,3,2-dioxaborolan-2-yl]-3-methylbutyl}amino)-2-oxoethyl]benzamideas a white solid (0.251 g, 93%). MS (m/z) in CH₃CN: [M+Et₃N+H]calculated for C₂₈H₄₅BCl₂N₃O₅, 584.3. found, 584.1. MS (m/z) in CH₃CN:[M−H] calculated for C₂₂H₂₈BCl₂N₂O₅, 481.1. found 481.1.

Example 5: Synthesis of2,5-dichloro-N-(2-{[(1R)-1-(4,4-dimethyl-5-oxo-1,3,2-dioxaborolan-2-yl)-3-methylbutyl]amino}-2-oxoethyl)benzamide(I-5)

To a solution of 2-hydroxyisobutyric acid (0.0567 g, 0.545 mmol) inEtOAc (2.0 mL) with an internal temperature of about 60° C. was added asolution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(0.200 g, 0.195 mmol) in EtOAc (1.0 mL). The solution was cooleduncontrolled until the internal temperature was about 25° C. and thesolvent was removed by evaporation to yield2,5-dichloro-N-(2-{[(1R)-1-(4,4-dimethyl-5-oxo-1,3,2-dioxaborolan-2-yl)-3-methylbutyl]amino}-2-oxoethyl)benzamideas a white solid (0.225 g, 96%). MS (m/z) in CH₃CN: [M+Et₃N+H]calculated for C₂₄H₃₉BCl₂N₃O₅, 530.2. found, 530.0. MS (m/z) in CH₃CN:[M−H] calculated for C₁₈H₂₂BCl₂N₂O₅, 427.1. found, 427.0.

Example 6: Synthesis of2,5-dichloro-N-[2-({(1R)-3-methyl-1-[(5R)-4-oxo-5-phenyl-1,3,2-dioxaborolan-2-yl]butyl}amino)-2-oxoethyl]benzamide(I-6)

To a solution of (R)-mandelic acid (0.168 g, 1.10 mmol) in EtOAc (2.0mL) with an internal temperature of about 60° C. was added a solution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(0.382 g, 0.37 mmol) in EtOAc (1.0 mL). The solution was cooleduncontrolled until the internal temperature was about 25° C. and theresulting precipitate was collected by filtration to yield2,5-dichloro-N-[2-({(1R)-3-methyl-1-[(5R)-4-oxo-5-phenyl-1,3,2-dioxaborolan-2-yl]butyl}amino)-2-oxoethyl]benzamideas a white solid (0.343 g, 65%). ¹H NMR (300 MHz, DMSO-d₆, δ): 10.88 (s,1H), 9.22 (m, 1H), 7.68-7.27 (m, 8H), 5.15 (s, 1H), 4.33 (d, J=6.0 Hz,2H), 2.8-2.76 (m, 1H), 1.71-1.62 (m, 1H), 1.50-1.28 (m, 2H), 0.89 (m,6H). MS (m/z) in CH₃CN: [M+Et₃N+H] calculated for C₂₈H₃₉BCl₂N₃O₅, 578.2.found, 578.1. MS (m/z) in CH₃CN: [M−H] calculated for C₂₂H₂₂BCl₂N₂O₅,475.1. found 475.1.

Example 7: Synthesis of2,5-dichloro-N-[2-({(1R)-3-methyl-1-[(4S)-4-methyl-5-oxo-1,3,2-dioxaborolan-2-yl]butyl}amino)-2-oxoethyl]benzamide(I-7)

To a solution of L-lactic acid (0.675 g, 7.34 mmol) in EtOAc (3.0 mL)with an internal temperature of about 70° C. was added a solution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(2.50 g, 2.43 mmol) in EtOAc (7.5 mL). The solution was cooleduncontrolled until the internal temperature was about 60° C. After 30min, heptane (11.5 mL) was added until the solution became turbid. Thesuspension was heated until the internal temperature was at or about 70°C., at which point a homogenous solution resulted. The solution wascooled at a rate of 0.17° C./min until the internal temperature wasabout 30° C., then cooled uncontrolled until the internal temperaturewas about 0° C. The resulting precipitate was collected by filtration toyield2,5-dichloro-N-[2-({(1R)-3-methyl-1-[(4S)-4-methyl-5-oxo-1,3,2-dioxaborolan-2-yl]butyl}amino)-2-oxoethyl]benzamideas a white, crystalline solid (2.32 g, 81%). MS (m/z) in CH₃CN:[M+Et₃N+H] calculated for C₂₃H₃₇BCl₂N₃O₅, 515.9. found, 516.0. MS (m/z)in CH₃CN: [M−H] calculated for C₁₇H₂₀BCl₂N₂O₅, 413.1. found 413.0.

The XRPD data for I-7 is shown in FIG. 5 and in Table 4.

TABLE 4 XRPD Data I-7 Angle 2-θ ° Intensity % 7.404 46 8.783 63.5 9.40216.1 11.9 20.6 12.195 100 13.71 7.3 14.594 26.5 15.302 8.3 15.772 3117.299 26.8 17.859 25.8 18.549 22.7 19.943 55.5 20.214 33.9 20.606 5021.48 15.6 21.887 23 22.75 30.1 23.028 53.1 23.334 28.9 24.243 18.2 25.213.3 25.566 37.7 27.221 10 29.103 9.2 29.383 12.6

Example 8: Synthesis of2,5-dichloro-N-[2-({(1R)-3-methyl-1-[(4S)-4-methyl-6-oxo-1,3,2-dioxaborinan-2-yl]butyl}amino)-2-oxoethyl]benzamide(I-8)

To a solution of (S)-3-hydroxybutyric acid (0.0598 g, 0.566 mmol) inEtOAc (2.0 mL) with an internal temperature of about 60° C. was added asolution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(0.200 g, 0.195 mmol) in EtOAc (1.0 mL). The solution was cooleduncontrolled until the internal temperature was about 25° C. and thesolvent was removed by evaporation to yield2,5-dichloro-N-[2-({(1R)-3-methyl-1-[(4S)-4-methyl-6-oxo-1,3,2-dioxaborinan-2-yl]butyl}amino)-2-oxoethyl]benzamideas a white solid (0.225 g, 95%). ¹H NMR (300 MHz, DMSO-d₆, δ): 10.45 (s,1H), 9.11 (t, J=6.0 Hz, 1H), 7.65 (m, 1H), 7.55 (m, 2H), 4.21 (d, J=6.0Hz, 2H), 3.98-3.90 (m, 1H), 2.51 (m, 1H), 2.33 (dd, J=19.2 Hz, J=2.7 Hz,1H), 2.24-2.21 (m, 1H), 1.61-1.52 (m, 1H), 1.33-1.19 (m, 2H), 1.07-1.04(m, 3H), 0.84 (m, 6H). MS (m/z) in CH₃CN: [M+Et₃N+H] calculated forC₂₄H₃₉BCl₂N₃O₅, 5302. found, 530.0. MS (m/z) in CH₃CN: [M−H] calculatedfor C₁₈H₂₂BCl₂N₂O₅, 427.1. found, 427.1.

Example 9: Synthesis of2,5-dichloro-N-(2-{[(1R)-1-(4,4-dimethyl-6-oxo-1,3,2-dioxaborinan-2-yl)-3-methylbutyl]amino}-2-oxoethyl)benzamide(I-9)

To a solution of β-hydroxyisovaleric acid (0.0841 g, 0.712 mmol) inEtOAc (2.0 mL) with an internal temperature of about 60° C. was added asolution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(0.260 g, 0.253 mmol) in EtOAc (1.0 mL). The solution was cooleduncontrolled until the internal temperature was about 25° C. and thesolvent was removed by evaporation to yield2,5-dichloro-N-(2-{[(1R)-1-(4,4-dimethyl-6-oxo-1,3,2-dioxaborinan-2-yl)-3-methylbutyl]amino}-2-oxoethyl)benzamideas a white solid (0.296 g, 95%). MS (m/z) in CH₃CN: [M+Et₃N+H]calculated for C₂₅H₄₁BCl₂N₃O₅, 544.3. found, 544.0. MS (m/z) in CH₃CN:[M−H] calculated for C₁₉H₂₄BCl₂N₂O₅, 441.1. found, 441.0.

Example 10: Synthesis of2,5-dichloro-N-[2-({(1R)-1-[(4S)-4-tert-butyl-5-oxo-1,3,2-dioxaborolan-2-yl]-3-methylbutyl}amino)-2-oxoethyl]-2,5-dichlorobenzamide(I-10)

To a solution of (S)-2-hydroxy-3,3-dimethylbutyric acid (0.0712 g, 0.553mmol) in EtOAc (2.0 mL) with an internal temperature of about 60° C. wasadded a solution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(0.200 g, 0.195 mmol) in EtOAc (1.0 mL). The solution was cooleduncontrolled until the internal temperature was about 25° C. and thesolvent was removed by evaporation to yield2,5-dichloro-N-[2-({(1R)-1-[(4S)-4-tert-butyl-5-oxo-1,3,2-dioxaborolan-2-yl]-3-methylbutyl}amino)-2-oxoethyl]-2,5-dichlorobenzamideas a white solid (0.245 g, 97%). MS (m/z) in CH₃CN: [M+Et₃N+H]calculated for C₂₆H₄₃BCl₂N₃O₅, 558.3. found, 558.0. MS (m/z) in CH₃CN:[M−H] calculated for C₂₀H₂₆BCl₂N₂O₅, 455.1. found, 455.0.

Example 11: Synthesis of2,5-dichloro-N-[2-({(1R)-1-[(4S)-4-isopropyl-5-oxo-1,3,2-dioxaborolan-2-yl]-3-methylbutyl}amino)-2-oxoethyl]benzamide(I-11)

To a solution of (S)-2-hydroxy-3-methylbutyric acid (0.0659 g, 0.558mmol) in EtOAc (2.0 mL) with an internal temperature of about 60° C. wasadded a solution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(0.200 g, 0.195 mmol) in EtOAc (1.0 mL). The solution was cooleduncontrolled until the internal temperature was about 25° C. and thesolvent was removed by evaporation to yield2,5-dichloro-N-[2-({(1R)-1-[(4S)-4-isopropyl-5-oxo-1,3,2-dioxaborolan-2-yl]-3-methylbutyl}amino)-2-oxoethyl]benzamideas a white solid (0.246 g, 99%). MS (m/z) in CH₃CN: [M+Na] calculatedfor C₁₉H₂₅BCl₂N₂NaO₅, 465.1. found, 465.1. MS (m/z) in CH₃CN: [M−H]calculated for C₁₉H₂₄BCl₂N₂O₅, 441.1. found, 441.0.

Example 12: Synthesis of2,5-dichloro-N-[2-({(1R)-1-[(4S)-4-isobutyl-5-oxo-1,3,2-dioxaborolan-2-yl]-3-methylbutyl}amino)-2-oxoethyl]benzamide(I-12)

To a solution of 2-hydroxyisocaproic acid (0.0752 g, 0.569 mmol) inEtOAc (2.0 mL) with an internal temperature of about 60° C. was added asolution ofN,N′N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(0.200 g, 0.195 mmol) in EtOAc (1.0 mL). The solution was cooleduncontrolled until the internal temperature was about 25° C. and thesolvent was removed by evaporation to yield2,5-dichloro-N-[2-({(1R)-1-[(4S)-4-isobutyl-5-oxo-1,3,2-dioxaborolan-2-yl]-3-methylbutyl}amino)-2-oxoethyl]benzamideas a white solid (0.253 g, 95%). MS (m/z) in CH₃CN: [M+Na] calculatedfor C₂₀H₂₇BCl₂N₂NaO₅, 479.1. found, 479.1. MS (m/z) in CH₃CN: [M−H]calculated for C₂₀H₂₆BCl₂N₂O₅, 455.1. found 455.1.

Example 13: Synthesis of2,5-dichloro-N-(2-{[(1R)-3-methyl-1-(4-oxo-4H-1,3,2-benzodioxaborinin-2-yl)butyl]amino}-2-oxoethyl)benzamide(I-13)

To a solution of salicylic acid (0.0758 g, 0.549 mmol) in EtOAc (2.0 mL)with an internal temperature of about 60° C. was added a solution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(0200 g, 0.195 mmol) in EtOAc (1.0 mL). The solution was cooleduncontrolled until the internal temperature was about 25° C. and theresulting precipitate was collected by filtration to yield2,5-dichloro-N-(2-{[(1R)-3-methyl-1-(4-oxo-4H-1,3,2-benzodioxaborinin-2-yl)butyl]amino}-2-oxoethyl)benzamideas a white solid (0.198 g, 78%). MS (m/z) in CH₃CN: [M+Na] calculatedfor C₂₁H₂₁BCl₂N₂NaO₅, 485.1. found, 485.1. MS (m/z) in CH₃CN: [M−H]calculated for C₂₁H₂₀BCl₂N₂O₅, 461.1. found, 461.0.

The XRPD data for I-13 is shown in FIG. 6 and in Table 5.

TABLE 5 XRPD Data I-13 Angle 2-θ ° Intensity % 6.784 88.1 8.372 10011.855 66.6 13.18 85.2 14.118 7.7 14.546 19.3 15.614 9.6 16.123 19.316.417 14.1 16.738 7.7 17.29 43.7 19.05 17.4 19.28 28.9 19.726 52.120.401 60.8 20.591 37.6 21.233 43.7 21.658 16.7 22.029 18.6 22.718 30.923.557 41.5 24.236 22.2 24.717 62.1 25.309 26 25.648 13.5 26.186 69.126.653 17.4 26.995 36.3 27.956 25.4 28.898 8.4 29.47 8.7

Example 14: Synthesis of2,5-dichloro-N-(2-{[(1R)-3-methyl-1-(5-oxo-4,4-diphenyl-1,3,2-dioxaborolan-2-yl)butyl]amino}-2-oxoethyl)benzamide(I-14)

To a solution of benzilic acid (0.126 g, 0.552 mmol) in EtOAc (2.0 mL)with an internal temperature of about 60° C. was added a solution ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)(0.200 g, 0.195 mmol) in EtOAc (1.0 mL). The solution was cooleduncontrolled until the internal temperature was about 25° C. and thesolvent was removed by evaporation to yield2,5-dichloro-N-(2-{[(1R)-3-methyl-1-(5-oxo-4,4-diphenyl-1,3,2-dioxaborolan-2-yl)butyl]amino}-2-oxoethyl)benzamideas a white solid (0.291 g, 95%). MS (m/z) in CH₃CN: [M+Na] calculatedfor C₂₈H₂₇BCl₂N₂NaO₅, 575.1. found, 575.2. MS (m/z) in CH₃CN: [M−H]calculated for C₂₈H₂₆BCl₂N₂O₅, 551.1. found, 551.1.

Example 15: Synthesis of2,2′-{2-[(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]-5-oxo-1,3,2-dioxaborolane-4,4-diyl}diaceticAcid (I-15)

To a solution of citric acid (0.257 g, 1.34 mmol) in EtOAc (7.4 mL) withan internal temperature of about 74° C. was addedN,N′,N″-(boroxin-2,4,6-triyltris{[(1R)-3-methylbutane-1,1-diyl]imino[(2S)-1-oxo-3-phenylpropane-1,2-diyl]})tripyrazine-2-carboxamide(0.500 g, 0.455 mmol) as a solid. The resulting solution was cooleduncontrolled until the internal temperature was about 25° C. and wasevaporated to yield2,2′-{2-[(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]-5-oxo-1,3,2-dioxaborolane-4,4-diyl}diaceticacid as a white solid (0.730 g, 99%). MS (m/z) in CH₃CN: [M+Et₃N+H]calcd for C₃₁H₄₅BN₅O₉, 642.3. found, 642.2. MS (m/z) in CH₃CN: [M−H]calcd for C₂₅H₂₈BN₄O₉, 539.2. found, 539.2.

Example 16: Synthesis ofN-[(1S)-1-benzyl-2-({(1R)-3-methyl-1-[(5R)-4-oxo-5-phenyl-1,3,2-dioxaborolan-2-yl]butyl}amino)-2-oxoethyl]pyrazine-2-carboxamide(I-16)

To a solution of (R)-mandelic acid (0.0738 g, 0.485 mmol) in EtOAc (2.0mL) with an internal temperature of about 60° C. was addedN,N′,N″-(boroxin-2,4,6-triyltris{[(1R)-3-methylbutane-1,1-diyl]imino[(2S)-1-oxo-3-phenylpropane-1,2-diyl]})tripyrazine-2-carboxamide(0.178 g, 0.162 mmol) as a solid. The solution was cooled uncontrolleduntil the internal temperature was about 25° C. and the resultingprecipitate was collected by filtration to yieldN-[(1S)-1-benzyl-2-({(1R)-3-methyl-1-[(5R)-4-oxo-5-phenyl-1,3,2-dioxaborolan-2-yl]butyl}amino)-2-oxoethyl]pyrazine-2-carboxamideas a white solid (0.195 g, 80%). MS (m/z) in CH₃CN: [M+Na] calculatedfor C₂₇H₂₉BN₄NaO₅, 523.2. found, 523.2. MS (m/z) in CH₃CN: [M−H]calculated for C₂₇H₂₈BN₄O₅, 499.2. found, 4992.

Example 17: Synthesis ofN-[(1S)-1-benzyl-2-({(1R)-3-methyl-1-[(5R)-4-oxo-5-phenyl-1,3,2-dioxaborolan-2-yl]butyl}amino)-2-oxoethyl]pyrazine-2-carboxamide(I-17)

To a solution of (S)-3-hydroxybutyric acid (0.0509 g, 0.489 mmol) inEtOAc (2.0 mL) with an internal temperature of about 60° C. was addedN,N′,N″-(boroxin-2,4,6-triyltris{[(1R)-3-methylbutane-1,1-diyl]imino[(2S)-1-oxo-3-phenylpropane-1,2-diyl]})tripyrazine-2-carboxamide(0.179 g, 0.163 mmol) as a solid. The solution was cooled uncontrolleduntil the internal temperature was about 25° C. and the solvent wasremoved by evaporation to yieldN-[(1S)-1-benzyl-2-({(1R)-3-methyl-1-[(4S)-4-methyl-6-oxo-1,3,2-dioxaborinan-2-yl]butyl}amino)-2-oxoethyl]pyrazine-2-carboxamideas a white solid (0213 g, 96%). MS (m/z) in CH₃CN: [M+Na] calculated forC₂₃H₂₉BN₄NaO₅, 475.2. found, 4752. MS (m/z) in CH₃CN: [M−H] calculatedfor C₂₃H₂₈BN₄O₅, 451.2. found, 451.1.

Example 18: Preparation of Formulations of4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicAcid (I-1) for Parenteral or Oral Administration

Formulation A. A vessel was charged with 90 mL water and citric acidmonohydrate (0.08 g) and sodium citrate dihydrate (1.5 g) were added andstirred until dissolved. To this solution,4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid (I-1) Form 2 (0.142 g) was added, and the mixture was stirred untila solution was obtained. To this solution, sodium chloride (0.45 g) wasadded, and the pH was adjusted to pH 5.45 using 2N HCl. The final volumeof the resulting solution was adjusted to 100 mL with water and filteredthrough a 0.2 μm PES membrane to yield Formulation A which was stored at−20° C.

Formulation B was prepared as for Formulation A, except that the pH wasadjusted to pH 6.2 using 2N NaOH.

Formulation C: A vessel was charged with 90 mL water and citric acidmonohydrate (0.08 g), sodium citrate dihydrate (1.5 g), and propyleneglycol (1.0 g) were added and stirred until dissolved. To this solution,4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid (I-1) Form 2 (0.142 g) was added, and the mixture was stirred untila solution was obtained. The pH was adjusted to 6.2 using 2N NaOH, andthe final volume of the resulting solution was adjusted to 100 mL withwater and filtered through a 0.2 μm PES membrane to yield Formulation Cwhich was stored at −20° C.

Example 19: In Situ Preparation of Formulations of4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicAcid (I-1) for Parenteral or Oral Administration

Stock Formulation Vehicle: A vessel was charged with approximately 160mL water and citric acid monohydrate (0.714 g) and sodium citratedihydrate (2.24 g) were added and stirred until dissolved. To thissolution, propylene glycol (2.0 g) was added, and the mixture wasstirred until a homogeneous solution was obtained. The final pH was pH5.14. The final weight of the resulting solution was adjusted to 200 g(assuming density of 1 g/mL) with water and filtered through a 0.2 m PESmembrane filter unit and stored at a temperature between about 2° C. andabout 8° C.

Formulation Stock (1 mg/mL): To a vessel, 0.105 grams (approximately95.4% purity) ofN,N′,N″-{boroxin-2,4,6-triyltris[[(1R)-3-methylbutane-1,1-diyl]imino(2-oxoethane-2,1-diyl)]}tris(2,5-dichlorobenzamide)was added. To this was added approximately 90 g of the Stock FormulationVehicle, and the resulting mixture was stirred for 48 hours protectedfrom light. The final pH was pH 5.12. The final weight of the resultingsolution was adjusted to 100 g (assuming density of 1 g/mL) with StockFormulation Vehicle and filtered through a 0.2 μm PES membrane filterunit and stored protected from light at a temperature between about 2°C. and about 8° C.

Formulation D: The Formulation Stock was diluted to concentrations of0.05 mg/mL and 0.1 mg/mL with Stock Formulation Vehicle prior to use.

Formulation E: The Formulation Stock was diluted to concentrations of0.05 mg/mL and 0.1 mg/mL with a 0.9% sodium chloride solution prior touse.

Example 20: 20S Proteasome Assay

To 1 μL of test compound dissolved in DMSO in a 384-well blackmicrotiter plate is added 25 μL of assay buffer at 37° C. containinghuman PA28 activator (Boston Biochem, 12 nM final) with Ac-WLA-AMC (β5selective substrate) (15 μM final), followed by 25 μL of assay buffer at37° C. containing human 20S proteasome (Boston Biochem, 0.25 nM final).Assay buffer is composed of 20 mM HEPES, 0.5 mM EDTA and 0.01% BSA,pH7.4. The reaction is followed on a BMG Galaxy plate reader (37° C.,excitation 380 nm, emission 460 nm, gain 20). Percent inhibition iscalculated relative to 0% inhibition (DMSO) and 100% inhibition (10 μMbortezomib) controls.

Example 21: Antiproliferation Assay

HCT-116 (1000) or other tumor cells in 100 μL of appropriate cellculture medium (McCoy's 5A for HCT-116, Invitrogen) supplemented with10% fetal bovine serum (Invitrogen) are seeded in wells of a 96-wellcell culture plate and incubated overnight at 37° C. Test compounds areadded to the wells and the plates are incubated for 96 hours at 37° C.MTT or WST reagent (10 μL, Roche) are added to each well and incubatedfor 4 hours at 37° C. as described by the manufacturer. For MTT themetabolized dye is solubilized overnight according to manufacturer'sinstructions (Roche). The optical density for each well is read at 595nm (primary) and 690 nm (reference) for the MTT and 450 nm for the WSTusing a spectrophotometer (Molecular Devices). For the MTT the referenceoptical density values are subtracted from the values of the primarywavelength. Percent inhibition is calculated using the values from aDMSO control set to 100%.

Example 22: In Vivo Tumor Efficacy Model

Freshly dissociated HCT-116 (2-5×10⁶) or other tumor cells in 100 μL ofRPMI-1640 media (Sigma-Aldrich) are aseptically injected into thesubcutaneous space in the right dorsal flank of female CD-1 nude mice(age 5-8 weeks, Charles River) using a 1 mL 26⅜-ga needle (BectonDickinson Ref#309625). Alternatively, some xenograft models require theserial passaging of tumor fragments. In these cases, small fragments oftumor tissue (approximately 1 mm³) are implanted subcutaneously in theright dorsal flank of anesthetized (3-5% isoflourane/oxygen mixture)C.B-17/SCID mice (age 5-8 weeks, Charles River) via a 13-ga trocar(Popper & Sons 7927). Beginning at day 7 after inoculation tumors aremeasured twice weekly using a vernier caliper. Tumor volumes arecalculated using standard procedures (0.5×(length×width)). When thetumors reach a volume of approximately 200 mm³ mice are randomized intotreatment groups and begin receiving drug treatment. Dosing andschedules are determined for each experiment based on previous resultsobtained from pharmacokinetic/pharmacodynamic and maximum tolerated dosestudies. The control group will receive vehicle without any drug.Typically, test compound (100-200 μL) is administered via intravenous(27-ga needle), oral (20-ga gavage needle) or subcutaneous (27-ganeedle) routes at various doses and schedules. Tumor size and bodyweight are measured twice a week and the study is terminated when thecontrol tumors reach approximately 2000 mm³.

Example 23: Synthesis ofN—((S)-1-((R)-3-methyl-1-(4-oxo-4H-benzo[d][1,3,2]dioxaborinin-2-yl)butylamino)-1-oxo-3-phenylpropan-2-yl)pyrazine-2-carboxamide(I-19)

A mixture ofN,N′,N″-(boroxin-2,4,6-triyltris{[(1R)-3-methylbutane-1,1-diyl]imino[(2S)-1-oxo-3-phenylpropane-1,2-diyl]})tripyrazine-2-carboxamide(0.250 g, 0.228 mmol) and salicyclic acid (269.6 mg, 0.68 mmol) weremixed in EtOAc (10 mL). The mixture was heated to form a solution. Thesolution was cooled uncontrolled until the internal temperature wasabout 25° C. Heptane (16 mL) was added. White solid precipitated out andthe resultant slurry was agitated at ambient temperature for 3 h. Theslurry was filtered to collect solidN—((S)-1-((R)-3-methyl-1-(4-oxo-4H-benzo[d][1,3,2]dioxaborinin-2-yl)butylamino)-1-oxo-3-phenylpropan-2-yl)pyrazine-2-carboxamide(0.249 g, 75%). MS (m/z) in CH₃CN: [M+H] calculated for C₂₆H₂₈BN₄O₅,487.2153. found, 487.3.

Example 24: Synthesis of2-((S)-2-((R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)propanamido)butyl)-5-oxo-1,3,2-dioxaborolan-4-yl)aceticAcid (I-20)

A mixture ofN,N′,N″-(boroxin-2,4,6-triyltris{[(1R)-3-methylbutane-1,1-diyl]imino[(2S)-1-oxo-3-phenylpropane-1,2-diyl]})tripyrazine-2-carboxamide(0.500 g, 0.455 mmol) and L-malic acid (213.6 mg, 0.55 mmol) were mixedin THF (5 mL). The mixture was heated to form a solution. The solutionwas cooled uncontrolled until the internal temperature was about 25° C.White solid precipitated out and the resultant slurry was agitated atambient temperature for 1 h. The slurry was filtered to collect solid2-((S)-2-((R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)propanamido)butyl)-5-oxo-1,3,2-dioxaborolan-4-yl)aceticacid (0.625 g, 95%). MS (m/z) in CH₃CN: [M+H] calculated forC₂₃H₂₈BN₄O₇, 483.2051. found, 4832.

Example 25: Synthesis of2-((R)-2-((R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)propanamido)butyl)-5-oxo-1,3,2-dioxaborolan-4-yl)aceticAcid (I-21)

A mixture ofN,N′N″-(boroxin-2,4,6-triyltris{[(1R)-3-methylbutane-1,1-diyl]imino[(2S)-1-oxo-3-phenylpropane-1,2-diyl]})tripyrazine-2-carboxamide(0.305 g, 0.278 mmol) and D-malic acid (130.3 mg, 0.33 mmol) were mixedin acetone (3 mL). The mixture was heated to form a solution. Thesolution was cooled uncontrolled until the internal temperature wasabout 25° C. White solid precipitated out and the resultant slurry wasagitated at ambient temperature for 3 h. The slurry was filtered tocollect solid2-((R)-2-((R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)propanamido)butyl)-5-oxo-1,3,2-dioxaborolan-4-yl)aceticacid (0.410 g, 100%). [M+H] calculated for C₂₃H₂₈BN₄O₇, 483.2051. found,483.2.

Example 26: Synthesis of(R)-2-hydroxy-2-((R)-2-((R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)propanamido)butyl)-5-oxo-1,3,2-dioxaborolan-4-yl)aceticAcid (I-22)

A mixture ofN,N′,N″-(boroxin-2,4,6-triyltris{[(1R)-3-methylbutane-1,1-diyl]imino[(2S)-1-oxo-3-phenylpropane-1,2-diyl]})tripyrazine-2-carboxamide(0.270 g, 0.246 mmol) and L-tartaric acid (149.5 mg, 0.33 mmol) weremixed in acetone (3 mL). The mixture was heated to form a solution. Thesolution was cooled uncontrolled until the internal temperature wasabout 25° C. Heptane (2.5 mL) was added. White solid precipitated outand the resultant slurry was agitated at ambient temperature for 1.5 h.The slurry was filtered to collect solid(R)-2-hydroxy-2-((R)-2-((R)-3-methyl-1-((S)-3-phenyl-2-(pyrazin-2-carboxamido)propanamido)butyl)-5-oxo-1,3,2-dioxaborolan-1-yl)aceticacid (0.388 g) which also contained a dimeric species. MS (m/z) inCH₃CN: [M+H] calculated for C₂₃H₂₈BN₄O₈, 4992000. found, 4992.

Example 27: Synthesis of(S)-2-hydroxy-2-((S)-2-((R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)propanamido)butyl)-5-oxo-1,3,2-dioxaborolan-4-yl)aceticAcid (I-23)

A mixture ofN,N′,N″-(boroxin-2,4,6-triyltris{[(1R)-3-methylbutane-1,1-diyl]imino[(2S)-1-oxo-3-phenylpropane-1,2-diyl]})tripyrazine-2-carboxamide(0.180 g, 0.164 mmol) and D-tartaric acid (147.5 mg, 0.33 mmol) weremixed in acetone (4 mL). The mixture was heated to form a solution. Thesolution was cooled uncontrolled until the internal temperature wasabout 25° C. Heptane (8 mL) was added. The mixture was evaporated toyield(S)-2-hydroxy-2-((S)-2-((R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)propanamido)butyl)-5-oxo-1,3,2-dioxaborolan-4-yl)aceticacid (0.447 g) which also contained a dimeric species. MS (m/z) inCH₃CN: [M+H] calculated for C₂₃H₂₈BN₄O₈, 499.2000. found, 499.2.

Example 28: Pharmaceutical Composition 1

The composition of the capsule is shown in Table 7 below.

TABLE 7 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.29 Microcrystalline cellulose (low moisture)Filler 89.71 Total capsule content weight, mg 90.00 Size 4 white opaquegelatin capsules

Example 29: Pharmaceutical Composition 2

The composition of the capsule is shown in Table 8 below.

TABLE 8 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.29 Silicified microcrystalline cellulose Filler109.71 Total capsule content weight, mg 110.00 Size 4 white opaquegelatin capsules

Example 30: Pharmaceutical Composition 3

The composition of the capsule is shown in Table 9 below.

TABLE 9 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.29 Microcrystalline cellulose (low moisture)Filler 88.81 Magnesium stearate Lubricant 0.90 Total capsule contentweight, mg 90.00 Size 4 white opaque gelatin capsule

Example 31: Pharmaceutical Composition 4

The composition of the capsule is shown in Table 10 below.

TABLE 10 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.29 Microcrystalline cellulose Filler 78.91Magnesium stearate Lubricant 0.80 Total capsule content weight, mg 80.00Size 4 white opaque gelatin capsule

Example 32: Pharmaceutical Composition 5

The composition of the capsule is shown in Table 11 below.

TABLE 11 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.29 Microcrystalline cellulose (low moisture)Filler 84.71 Total capsule contents weight, mg 85.00 Size 4 white opaquegelatin capsule

Example 33: Pharmaceutical Composition 6

The composition of the capsule is shown in Table 12 below.

TABLE 12 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.72 Microcrystalline cellulose (low moisture)Filler 119.28 Total capsule content weight, mg 120.00 Size 3 dark greengelatin capsule

Example 34: Pharmaceutical Composition 7

The composition of the capsule is shown in Table 13 below.

TABLE 13 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 2.89 Microcrystalline cellulose (low moisture)Filler 147.11 Total capsule content weight, mg 150.00 Size 2 Swedishorange gelatin capsule

Example 35: Pharmaceutical Composition 8

The composition is shown in Table 14 below.

TABLE 14 Batch composition Item Number Component g/Batch mg/Capsule 1Compound of formula (I-1) Form 2 7.06 0.30 2 Microcrystalline cellulose,NF 100 4.25 (Emcocel ® XLM90; low moisture) 3 Microcrystallinecellulose, NF 192.9 8.20 (Emcocel ® XLM90; low moisture) 4Microcrystalline cellulose, NF 300 12.75 (Emcocel ® XLM90; low moisture)5 Microcrystalline cellulose, NF 500 21.25 (Emcocel ® XLM90; lowmoisture) 6 Microcrystalline cellulose, NF 900 38.25 (Emcocel ® XLM90;low moisture) Total weight 2000.0 85.00 Size 4 white opaque gelatincapsules

The batch was prepared according to the following process:

-   -   1) Microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #2) was screened through a 40 micron mesh screen.    -   2) The screened material from step 1) was added to the PK        blender and was blended for 2 minutes.    -   3) Compound of formula (I-1) Form 2 that had been screened        through a 60 micron mesh screen, was weighed out (Item #1).    -   4) The compound of formula (I-1) Form 2 from step 3), and the        microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #3) were combined in a polyethylene bag, and the        polyethylene bag was shaken; then the contents of the        polyethylene bag were passed through the same 40 micron screen        as was used in step 1).    -   5) The material from step 4) was added to the PK blender and        blended for 15 minutes.    -   6) Microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #4) was screened through the same 40 micron mesh screen,        transferred to the same polyethylene bag used in step 4) and        shaken in the polyethylene bag.    -   7) The material from step 6) was added to the PK blender, which        still contained the material from step 5) and blended for 10        minutes.    -   8) Microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #5) was screened through the same 40 micron mesh screen,        transferred to the same polyethylene bag used in steps 4) and        6), and shaken in the polyethylene bag.    -   9) The material from step 8) was added to the PK blender, which        still contained the material from steps 5) and 7), and blended        for 10 minutes.    -   10) Microcrystalline cellulose, NF (Emcocel® XLM90; low        moisture) (Item #6) was screened through the same 40 micron mesh        screen, transferred to the same polyethylene bag used in steps        4), 6), and 8), and shaken in the polyethylene bag.    -   11) The material from step 10) was added to the PK blender,        which still contained the material from steps 5), 7), and 9),        and blended for 10 minutes.    -   12) The material from the blender was encapsulated in size 4        white opaque gelatin capsules using the In-Cap system.    -   13) The capsules were de-dusted, and weight-sorted.

Example 36: Pharmaceutical Composition 9

The composition of the capsules is shown in Table 15 below.

TABLE 15 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.3 Pregeletanized Starch (Starch 1500) Filler122.825 Talc Flow-aid 1.25 Magnesium Stearate Lubricant 0.625 Totalcapsule content weight, mg 125.00 Size 4 white opaque gelatin capsule

Example 37: Pharmaceutical Composition 10

The composition of the capsules is shown in Table 16 below.

TABLE 16 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.3 Pregeletanized Starch (Starch 1500) Filler124.7 Total capsule content weight, mg 125.00 Size 4 white opaquegelatin capsule

Example 38: Pharmaceutical Composition 11

The composition of the capsule is shown in Table 17 below.

TABLE 17 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.3 Microcrystalline cellulose Filler 124.7(Emcocel ® XLM90; low moisture) Talc Flow-aid 1.25 Total capsule contentweight, mg 125.00 Size 4 white opaque gelatin capsule

Example 39: Pharmaceutical Composition 12

The composition of the capsule is shown in Table 18 below.

TABLE 18 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.3 Microcrystalline cellulose Filler 89.25(Emcocel ® XLM90; low moisture) Magnesium Stearate Lubricant 0.45 Totalcapsule content weight, mg 90.00 Size 4 white opaque gelatin capsule

Example 40: Pharmaceutical Composition 13

The composition of the capsule is shown in Table 19 below.

TABLE 19 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.3 Microcrystalline cellulose Filler 88.35(Emcocel ® XLM90; low moisture) Talc Flow-aid 0.9 Magnesium StearateLubricant 0.45 Total capsule content weight, mg 90.00 Size 4 whiteopaque gelatin capsule

Example 41: Pharmaceutical Composition 14

The composition of the capsule is shown in Table 20 below.

TABLE 20 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.3 Microcrystalline cellulose Filler 51.15(Emcocel ® XLM90; low moisture) Talc Flow-aid 0.98 Magnesium StearateLubricant 0.49 Pregeletanized Starch (Starcap) 45.08 Total capsulecontent weight, mg 98.00 Size 4 white opaque gelatin capsule

Example 42: Pharmaceutical Composition 15

The composition of the capsule is shown in Table 21 below.

TABLE 21 Capsule composition Component Function mg/Capsule Compound offormula (I-1) Form 2 0.3 Microcrystalline cellulose Filler 61.65(Emcocel ® XLM90; low moisture) Talc Flow-aid 1.18 Magnesium StearateLubricant 0.59 Sodium Starch Glycolate (Explotab) 54.28 Total capsulecontent weight, mg 118.00 Size 4 white opaque gelatin capsule

Example 43: Pharmaceutical Composition 16

The composition of the batch is shown in Table 22 below.

TABLE 22 Batch composition Item Number Component g/Batch mg/Capsule 1Compound of formula (I-1) Form 2 0.33 0.30 2 Microcrystalline cellulose,NF 5.00 4.50 (Emcocel ® XLM90; low moisture) 3 Microcrystallinecellulose, NF 8.17 7.35 (Emcocel ® XLM90; low moisture) 4Microcrystalline cellulose, NF 14.00 12.60 (Emcocel ® XLM90; lowmoisture) 5 Microcrystalline cellulose, NF 25.00 22.50 (Emcocel ® XLM90;low moisture) 6 Microcrystalline cellulose, NF 44.00 39.60 (Emcocel ®XLM90; low moisture) 7 Talc 1.00 0.90 8 Sodium Citrate 2.00 1.80 9Magnesium Stearate 0.50 0.45 Total weight 100.00 90.00 Size 4 whiteopaque gelatin capsules

The batch was prepared according to the following process:

-   -   1) Microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #2) was screened through a 40 micron mesh screen.    -   2) The screened material from step 1) was added to the PK        blender and was blended for 2 minutes.    -   3) Compound of formula (I-1) Form 2 that had been screened        through a 60 micron mesh screen, was weighed out (Item #1).    -   4) The compound of formula (I-1) Form 2 from step 3), and the        microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #3) were combined in a polyethylene bag, and the        polyethylene bag was shaken; then the contents of the        polyethylene bag were passed through the same 40 micron screen        as was used in step 1).    -   5) The material from step 4) was added to the PK blender and        blended for 15 minutes.    -   6) Microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #4) was screened through the same 40 micron mesh screen,        transferred to the same polyethylene bag used in step 4) and        shaken in the polyethylene bag.    -   7) Talc (Item #7), and Sodium Citrate (Item #8) were screened        through the same 40 micron mesh screen.    -   8) The materials from step 6) and 7) were added to the PK        blender, which still contained the material from step 5) and        blended for 10 minutes.    -   9) Microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #5) was screened through the same 40 micron mesh screen,        transferred to the same polyethylene bag used in steps 4) and        6), and shaken in the polyethylene bag.    -   10) The material from step 9) was added to the PK blender, which        still contained the material from steps 5) and 8), and blended        for 10 minutes.    -   11) Microcrystalline cellulose, NF (Emcocel® XLM90; low        moisture) (Item #6) was screened through the same 40 micron mesh        screen, transferred to the same polyethylene bag used in steps        4), 6), and 9), and shaken in the polyethylene bag.    -   12) The material from step 11) was added to the PK blender,        which still contained the material from steps 5), 8), and 10),        and blended for 10 minutes.    -   13) Magnesium Stearate (Item #9) was screened through the same        40 micron mesh screen.    -   14) The material from step 13) was added to the PK blender,        which still contained the material from steps 5), 8), 10),        and 12) and blended for 5 minutes.    -   15) The material from the blender was encapsulated in size 4        white opaque gelatin capsules using the Profill system.    -   16) The capsules were de-dusted, and weight-sorted.

Example 44: Pharmaceutical Composition 17

The composition of the batch is shown in Table 23 below.

TABLE 23 Batch composition Item Number Component g/Batch mg/Capsule 1Compound of formula (I-1) Form 2 7.06 0.30 2 Microcrystalline cellulose,NF 4.94 0.21 (Emcocel ® XLM90; low moisture) 3 Microcrystallinecellulose, NF 25.00 1.06 (Emcocel ® XLM90; low moisture) 4Microcrystalline cellulose, NF 53.00 2.25 (Emcocel ® XLM90; lowmoisture) 5 Talc 10.00 0.43 6 Microcrystalline cellulose, NF 90 3.83(Emcocel ® XLM90; low moisture) 7 Talc 30 1.28 8 Microcrystallinecellulose, NF 170 7.23 (Emcocel ® XLM90; low moisture) 9Microcrystalline cellulose, NF 300 12.75 (Emcocel ® XLM90; low moisture)10 Microcrystalline cellulose, NF 500 21.25 (Emcocel ® XLM90; lowmoisture) 11 Microcrystalline cellulose, NF 800 34 (Emcocel ® XLM90; lowmoisture) 12 Magnesium Stearate 10 0.43 Total weight 2000 85 Size 4white opaque gelatin capsules

The batch was prepared according to the following process:

-   -   1) Microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #2) was screened through a 40 micron mesh screen.    -   2) The screened material from step 1) was added to the small PK        blender and blended for 2 minutes.    -   3) Compound of formula (I-1) Form 2 that had been screened        through a 60 micron mesh screen was weighed out (Item #1).    -   4) The compound of formula (I-1) Form 2 from step 3), and the        microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #3) were combined, and then passed through the same 40        micron screen as was used in step 1).    -   5) The material from step 4) was added to the small PK blender        and blended for 30 minutes.    -   6) Microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #4) and Talc (Item #5) were screened through the same 40        micron mesh screen.    -   7) The material from step 6) was added to the small PK blender,        which still contained the material from step 5) and blended for        15 minutes.    -   8) Microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #6) was screened through the same 40 micron mesh screen,        transferred to a second larger PK blender, and blended for 2        minutes.    -   9) The contents of the small PK blender from steps 5) and 7)        were emptied into a polyethylene bag, and then transferred to        the larger PK blender from step 8).    -   10) Talc (Item #7) and Microcrystalline cellulose, NF (Emcocel®        XLM90; low moisture) (Item #8) were screened through the same 40        micron mesh screen.    -   11) Half the material from step 10) was added to the small PK        blender from steps 5) and 7), blended for 3 minutes, transferred        to the same polyethylene bag used in step 9), and shaken in the        polyethylene bag.    -   12) The material from step 11) was added to the larger PK        blender, which still contained the material from steps 8) and        9).    -   13) The second half of material from step 10) was added to the        small PK blender from steps 5) and 7), and 11), blended for 3        minutes, transferred to the same polyethylene bag used in        steps 9) and 11), and shaken in the polyethylene bag.    -   14) The material from step 13) was added to the larger PK        blender, which still contained the material from steps 8), 9),        and 12), and blended for 10 minutes.    -   15) Microcrystalline cellulose, NF (Emcocel® XLM90; low        moisture) (Item #9) was screened through the same 40 micron mesh        screen, transferred to the same polyethylene bag used in steps        9), 11), and 13), and shaken in the polyethylene bag.    -   16) The material from step 15) was added to the same larger PK        blender, which still contained material from steps 8), 9), 12),        and 14), and blended for 10 minutes.    -   17) Microcrystalline cellulose, NF (Emcocel® XLM90; low        moisture) (Item #10) was screened through the same 40 micron        mesh screen.    -   18) The material from step 17) was added to the same larger PK        blender, which still contained material from steps 8), 9), 12),        14), and 16), and blended for 10 minutes.    -   19) Microcrystalline cellulose, NF (Emcocel® XLM90; low        moisture) (Item #11) was screened through the same 40 micron        mesh screen.    -   20) The material from step 19) was added to the same larger PK        blender, which still contained material from steps 8), 9), 12),        14), 16), and 18), and blended for 10 minutes.    -   21) Magnesium Stearate (Item #12) was screened through the same        40 micron mesh screen.    -   22) The material from step 21) was added to the same larger PK        blender, which still contained material from steps 8), 9), 12),        14), 16), 18), and 20), and blended for 5 minutes.    -   23) The material from the blender was encapsulated in size 4        white opaque gelatin capsules using the Incap system.    -   24) The capsules were de-dusted, and weight-sorted.

Example 45: Pharmaceutical Composition 18

The composition of the batch is shown in Table 24 below.

TABLE 24 Batch composition Item Number Component g/Batch mg/Capsule 1Compound of formula (I-1) Form 2 3.53 0.30 2 Microcrystalline cellulose,NF 496.5 4.50 (Emcocel ® XLM90; low moisture) 3 Microcrystallinecellulose, NF 500 7.35 (Emcocel ® XLM90; low moisture) Total weight 100085 Size 4 white opaque gelatin capsules

The batch is prepared according to the following process:

-   -   1) Microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #2) is screened through a 40 micron mesh screen and added        to a high shear mixer.    -   2) Compound of formula (I-1) Form 2 is screened through a 60        micron mesh screen and weighed out (Item #1) and added to the        same high shear mixer from step 1).    -   3) Microcrystalline cellulose, NF (Emcocel® XLM90; low moisture)        (Item #3) is screened through the same 40 micron mesh screen,        and added to the same high shear mixer from steps 1) and 2).    -   4) The high shear mixer from steps 1), 2), and 3) is run for 4        minutes.    -   5) The material from the high shear mixer is encapsulated in        size 4 white opaque gelatin capsules using the Incap system.    -   6) The capsules are de-dusted, and weight-sorted.

Example 46: Lyophilized Powder 1

In a clean container, a solution of 40% tert-butyl alcohol/60% water forinjection was prepared by warming the required amount of tert-butylalcohol to 35° C. and adding water for injection. The solution wascooled to 15-30° C. A portion of the required amount (60% of the totalbatch) of tert-butyl alcohol/water solution was added to apre-compounding container. Approximately 40% of the solution wasreserved for use in rinsing. Citric acid (30% of the batch amount) wasadded to the pre-compounding container with stirring. The container wasrinsed with the reserved tert-butyl alcohol/water solution, and therinses were added to the pre-compounding container. The mixture wasstirred until the citric acid was completely dissolved. Sodium citrate(30% of the batch amount) was added to the pre-compounding containerwith stirring. The container was rinsed with the reserved tert-butylalcohol/water solution, and the rinses were added to the pre-compoundingcontainer. The mixture was stirred until the sodium citrate wascompletely dissolved. N-(2-pyrazine)carbonyl-L-phenyl-L-leucine boronicacid (VIII-15) was added to the pre-compounding container with stirring.The container was rinsed with the reserved tert-butyl alcohol/watersolution, and the rinses were added to the pre-compounding container.The mixture was stirred until the boronic acid was completely dissolved.The citric acid, sodium citrate, and boronic acid mixture from thepre-compounding container was transferred to the main compoundingvessel. The pre-compounding container was rinsed with water forinjection and the rinses were added to the main compounding vessel.Citric acid (70% of the batch amount) was added to the main vessel withstirring. The container was rinsed with the water, and the rinses wereadded to the main vessel. The mixture was stirred until the citric acidwas completely dissolved. Sodium citrate (70% of the batch amount) wasadded to the main vessel with stirring. The container was rinsed withwater, and the rinses were added to the pre-compounding container. Themixture was stirred until the sodium citrate was completely dissolved.Glycine was added to the main vessel and the residual glycine was rinsedwith water, and the rinses were added to the main vessel. The mixturewas stirred until the glycine was completely dissolved. Sufficient waterwas added to reduce the total alcohol content to 4.7% v/v. The mixturewas filtered through a 0.22 μm filter. Aliquots of the filtered solutionwere placed into vials. The vials were sealed with lyophilizationstoppers and were placed on lyophilizer chamber shelves maintained at20° C. The lyophilization chamber shelves were cooled to −45° C. usingthe appropriate ramp rate and held at that temperature for 200 minutes.The shelf was warmed to −20° C. using an appropriate ramp rate andmaintained at that temperature for 480 minutes. The shelf was re-cooledto −45° C. using an appropriate ramp rate and maintained at thattemperature. After 200 minutes, the lyophilization chamber wasevacuated, and the chamber pressure was adjusted to 150 microns withnitrogen. The chamber shelves were warmed up to −25° C. using anappropriate ramp rate, and held at that temperature for 3000 minutes.After each of the product thermocouples read −25° C. or warmer, theshelf was warmed to 27° C. and maintained at that temperature for 600minutes. At the end of the terminal drying phase, the chamber pressurewas restored using nitrogen, and vials were scaled and removed. Prelyophilized solution contained: 52 mM citrate, 3% glycine, 4.7%tert-butyl alcohol (as shown in Table 25 below).

TABLE 25 Batch composition Amount No. Component Amount/mL mM Batch pervial 1. Compound (VIII-15) 0.001 g 2.6 0.300 g 3.5 mg 2. Citric AcidMonohydrate, USP/EP 0.00382 g 18.2 1.147 g 13.37 mg 3. Sodium CitrateDihydrate, USP/EP 0.00994 g 33.8 2.982 g 34.79 mg 4. Glycine, USP/EP0.03 g 399.6 9.0 g 105 mg 5. Tert-butyl alcohol, ACS n/a n/a 14.1 mL0.1645 mL 6. Water for Injection, USP/EP n/a n/a Fill to n/a batchvolume 7. Total volume n/a n/a 300 mL 3.5 mL 8. Final measured pH n/an/a 5.08 n/a

Example 47: Lyophilized Powder 2

Prepared as described in Example 46. Pre-lyophilized solution contained:52 mM citrate; 3% glycine; and 4.7% tert-butyl alcohol (as shown inTable 26 below).

TABLE 26 Batch composition Amount No. Component Amount/mL mM Batch pervial 1. Compound (VIII-15) 0.001 g 2.6 0.300 g 3.5 mg 2. Citric AcidMonohydrate, USP/EP 0.00168 g 8.0 0.504 g 5.88 mg 3. Sodium CitrateDihydrate, USP/EP 0.0129 g 44.0 3.882 g 45.15 mg 4. Glycine, USP/EP 0.03g 399.6 9.0 g 105 mg 5. Tert-butyl alcohol, ACS grade n/a n/a 14.1 mL0.1645 mL 6. Water for Injection, USP/EP n/a n/a Fill to n/a batchvolume 7. Total volume n/a n/a 300 mL 3.5 mL 8. Final measured pH n/an/a 5.84 n/a

Example 48: Lyophilized Powder 3

The formulation was prepared as described in Example 46, except that thelyophilization cycle was modified. The vials were sealed withlyophilization stoppers and placed on lyophilizer chamber shelvesmaintained at 20° C. The lyophilization chamber shelves were cooled to−45° C. using the appropriate ramp rate and held at that temperature for200 minutes. The shelf was warmed to −20° C. using an appropriate ramprate and maintained at that temperature for 480 minutes. The shelf wasre-cooled to −45° C. using an appropriate ramp rate and maintained atthat temperature. After 200 minutes, the lyophilization chamber wasevacuated, and the chamber pressure was adjusted to 150 microns withnitrogen. The chamber shelves was warmed up to −15° C. using anappropriate ramp rate, and held at that temperature for 2700 minutes.After each of the product thermocouples read −15° C. or warmer, theshelf was warmed to 37° C. and maintained at that temperature for 300minutes. At the end of the terminal drying phase, the chamber pressurewas restored using nitrogen, and the vials were sealed and removed.Pre-lyophilized solution contained: 52 mM citrate; 3% glycine; and 4.7%tert-butyl alcohol (as shown in Table 27 below).

TABLE 27 Batch composition Amount No. Component Amount/mL mM Batch pervial 1. Compound (VIII-15) 0.001 g 2.6 1.0 g 3.5 mg 2. Citric AcidMonohydrate, USP/EP 0.00382 g 18.2 3.82 g 13.37 mg 3. Sodium CitrateDihydrate, USP/EP 0.00994 g 33.8 9.94 g 34.79 mg 4. Glycine, USP/EP 0.03g 399.6 30.0 g 105 mg 5. Tert-butyl alcohol, ACS grade n/a n/a 47.0 mL0.1645 mL 6. Water for Injection, USP/EP n/a n/a Fill to n/a batchvolume 7. Total volume n/a n/a 1000 mL 3.5 mL 8. Final measured pH n/an/a 5.05 n/a

Example 49: Lyophilized Powder 4

A clean vessel was charged with water for injection. Citric acid andsodium citrate were added and stirred until dissolved. To this solution,N-(2-pyrazine)carbonyl-L-phenyl-L-leucine boronic acid (VIII-15) wasadded and stirred until dissolved. Glycine was added to the vessel andthe residual glycine was rinsed with water, and the rinses were added tothe main vessel. The mixture was stirred until the glycine wascompletely dissolved. Sufficient water was added to batch volume. Themixture was filtered through a 0.22 μm filter. Aliquots of the filteredsolution were placed into vials. The vials were sealed withlyophilization stoppers and were placed on lyophilizer chamber shelvesmaintained at 20° C. The lyophilization chamber shelves were cooled to−45° C. using the appropriate ramp rate and held at that temperature for200 minutes. The shelf was warmed to −20° C. using an appropriate ramprate and maintained at that temperature for 480 minutes. The shelf wasre-cooled to −45° C. using an appropriate ramp rate and maintained atthat temperature. After 200 minutes, the lyophilization chamber wasevacuated with and the chamber pressure was adjusted to 150 microns withnitrogen. The chamber shelves were warmed up to −25° C. using anappropriate ramp rate, and held at that temperature for 3000 minutes.After each of the product thermocouples read −25° C. or warmer, theshelf was warmed to 27° C. and maintained at that temperature for 600minutes. At the end of the terminal drying phase, the chamber pressurewas restored using nitrogen, and vials were sealed and removed.Pre-lyophilized solution contained: 52 mM citrate; and 3% glycine (asshown in Table 28 below).

TABLE 28 Batch composition Amount Component Amount/mL mM Batch pervial 1. Compound (VIII-15) 0.001 g 2.6 0.30 g 3.5 mg 2. Citric AcidMonohydrate, USP/EP 0.004097 g 19.5 1.229 g 14.34 mg 3. Sodium CitrateDihydrate, USP/EP 0.009557 g 32.5 2.867 g 33.45 mg 4. Glycine, USP/EP0.03 g 399.6 9.0 g 105 mg 5. Water for Injection, USP/EP n/a n/a Fill ton/a batch volume 6. Total volume n/a n/a 300 mL 3.5 mL 7. Final measuredpH n/a n/a 4.90 n/a

Example 50: Lyophilized Powder 5

Prepared as described in Example 49. Pre-lyophilized solution contained:52 mM citrate; and 3% glycine (as shown in Table 29 below). In thisexample, the pH of the pre-lyophilized solution was adjusted to thefinal measured pH by the addition of 2N HCl.

TABLE 29 Batch composition Amount No. Component Amount/mL mM Batch pervial 1. Compound (VIII-15) 0.001 g 2.6 0.30 g 3.5 mg 2. Citric AcidMonohydrate, USP/EP 0.00168 g 8.0 0.504 g 5.88 mg 3. Sodium CitrateDihydrate, USP/EP 0.01294 g 44.0 3.882 g 45.29 mg 4. Glycine, USP/EP0.03 g 399.6 9.0 g 105 mg 5. Water for Injection, USP/EP n/a n/a Fill ton/a batch volume 6. Total batch volume n/a n/a 300 mL 3.5 mL 7. Finalmeasured pH n/a n/a 5.84 n/a

Example 50: Lyophilized Powder 6

A clean vessel is charged with water for injection. Citric acid andsodium citrate are added and stirred until dissolved. To this solution,4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid (I-1) is added and stirred until dissolved. Glycine is added to thevessel and the residual glycine is rinsed with water, and the rinses areadded to the main vessel. The mixture is stirred until the glycine iscompletely dissolved. Sufficient water is added to batch volume. Themixture is filtered through a 0.22 μm filter. Aliquots of the filteredsolution are placed into sterilized vials. The vials are sealed withlyophilization stoppers and are placed on lyophilizer chamber shelvesmaintained at 20° C. The lyophilization chamber shelves are cooled to−45° C. using an appropriate ramp rate, and then are held at thattemperature for 200 minutes. The shelf is warmed to −20° C. using anappropriate ramp rate and then is maintained at that temperature for 480minutes. The shelf is re-cooled to −45° C. using an appropriate ramprate and is maintained at that temperature. After 200 minutes, thelyophilization chamber is evacuated, and the chamber pressure isadjusted to 150 microns with nitrogen. The chamber shelves are warmed upto −25° C. using an appropriate ramp rate, and held at that temperaturefor 3000 minutes. After each of the product thermocouples reads −25° C.or warmer, the shelf is warmed to 27° C. and is maintained at thattemperature for 600 minutes. At the end of the terminal drying phase,the chamber pressure is restored using nitrogen, and vials are sealedand removed. The composition of the pre-lyophilized solution is 55 mMcitrate; and 3% glycine (as shown in Table 30 below).

TABLE 30 Batch composition Amount No. Component Amount/mL mM Batch pervial 1. Compound (I-1) [expressed as 0.001 g 2.75 0.50 g 3.5 mg amountof compound (VIII-1)] 2. Citric Acid Monohydrate, USP/EP 0.0012 g 5.50.578 g 4.2 mg 3. Sodium Citrate Dihydrate, USP/EP 0.0147 g 49.5 7.279 g51.45 mg 4. Glycine, USP/EP 0.03 g 399.6 15.0 g 105 mg 5. Water forInjection, USP/EP n/a n/a Fill to n/a batch volume 6. Total volume n/an/a 500 mL 3.5 mL

Example 51: Reconstitution of Lyophilized Powders

The lyophilized powders (e.g. as prepared in Examples 46-50) areanalyzed using XRPD, DSC, gas chromatography, and Karl Fisher for cakestructure, cake stability, residual solvent, and residual moisture,respectively. The lyophilized powders are reconstituted with theappropriate amount of sterile water for injection or with sterile 0.9%sodium chloride solution for injection. The reconstituted solutions areanalyzed using HPLC, and NMR, for purity, and percentage ester.

Example 52: Preparation of Formulation of4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicAcid (I-1) Form 2 for Parenteral or Oral Administration

A vessel was charged with water and citric acid monohydrate and sodiumcitrate dihydrate were added and stirred until dissolved. To thissolution,4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid (I-1) Form 2 was added, and the mixture was stirred until asolution was obtained. To this solution, sodium chloride was added andstirred until dissolved. Sufficient water was added to batch volume andthe solution was filtered through a 0.2 μm PES membrane. Aliquots of thefiltered solution were placed into vials. The vials were sealed withstoppers and stored at −20° C. The batch and vial composition is asdescribed below in Table 31.

TABLE 31 Batch composition Amount No. Component Amount/mL mM Batch pervial 1. Compound (I-1) [expressed 0.001 g 2.75 20 g 3.3 mg as amount ofcompound (VIII-1)] 2. Citric Acid Monohydrate, USP/EP 0.0012 g 5.523.198 g 3.282 mg 3. Sodium Citrate Dihydrate, USP/EP 0.0147 g 49.5291.183 g 48.05 mg 4. Sodium Chloride, USP/EP 0.0045 g 77 89.991 g 14.85mg 5. Water for Injection, USP/EP n/a n/a Fill to n/a batch volume 6.Total volume n/a n/a 20 L 3.3 mL 7. Final measured pH n/a n/a 5.72 n/a

Example 53: Analytical Test Method 1

Reversed-phase HPLC using a C8 column at 25° C. with ultraviolet (UV)detection at 225 nm.

Mobile phase: The gradient system starts at 85% mobile phase A (0.01%trifluoroacetic acid in water) and 15% mobile phase B (0.01%trifluoroacetic acid in acetonitrile) and ends at 75% mobile phase Bafter 40 minutes.

The test sample is prepared by dissolving the content of the capsules indiluent which is 15:85 (v/v) acetonitrile:20 mM citrate buffer. Underthese aqueous conditions, the compound of formula (I-1) completelyhydrolyzes the citrate ester portion of the molecule to give thecompound of formula (VIII-1) in a 1:1 molecular ratio. The presence ofthe compound of formula (VIII-I) in the test sample is confirmed bycomparison of the sample retention time to that of the referencestandard. The amount of the compound of formula (VIII-1) present in asample is calculated from the area under the peak, on a weight-to-weightcomparison including molecular weight conversion, with the area underthe peak of the reference standard. The reference standard employed is aknown amount of the compound of formula (I-1), of known purity, which isprepared under the same hydrolyzing conditions as the test sample. Thelimit of quantitation for the method is 0.05% and the calculated limitof detection is 0.02%.

Example 54: Analytical Test Method 2

Normal-phase HPLC using isocratic elution with a mobile phase of40/60/0.1 (v/v/v) THF/n-Hexane/TFA on a cyano HPLC column at 25° C. for8 minutes, with UV detection at 230 nm.

The test sample is prepared by dissolving the content of the capsules in40/60 (v/v) THF/n-Hexane. Under these conditions, the compound offormula (I-1) is not hydrolyzed to the compound of formula (VIII-1). Theamount of the compound of formula (VIII-1) present in the test sample iscalculated from the area under the peak, on a weight to weightcomparison, with the area under the peak of the reference standard. Thereference standard employed is a known amount of the compound of formula(VIII-1), of known purity, which is prepared under the same conditionsas the test sample. The limit of quantitation for detection of thecompound of formula (I-1) is 0.2%.

To calculate the amount of compound of formula (I-1) present in a testsample, both Analytical Test Method 1 and Analytical Test Method 2 areused. Analytical Test Method 1 is used to calculate the amount on aweight basis of the compound of formula (VIII-1) that is present in atest sample, containing the compound of formula (I-1). Analytical TestMethod 2 is also used to calculate the amount of the compound of formula(VIII-1) present in the sample of the compound of formula (I-1) obtainedwithout induced hydrolysis.

The amount of the compound of formula (VIII-1) obtained from AnalyticalTest Method 1 minus the amount of the compound of formula (VIII-1)obtained from Analytical Test Method 2 gives the measured amount of thecompound of formula (VIII-1) that is produced by the induced hydrolysisof the compound of formula (I-1) present in the test sample. Based on a1:1 molecular ratio, a molecular weight calculation gives the amount ofthe compound of formula (I-1) present in the test sample.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, these particular embodiments areto be considered as illustrative and not restrictive. It will beappreciated by one skilled in the art from a reading of this disclosurethat various changes in form and detail can be made without departingfrom the true scope of the invention, which is to be defined by theappended claims rather than by the specific embodiments.

The patent and scientific literature referred to herein establishesknowledge that is available to those with skill in the art. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. The issued patents, applications,and references that are cited herein are hereby incorporated byreference to the same extent as if each was specifically andindividually indicated to be incorporated by reference. In the case ofinconsistencies, the present disclosure, including definitions, willcontrol.

What is claimed is:
 1. A process for generating a compound of Formula (II)

or a pharmaceutically acceptable salt thereof, wherein A is 0, 1, or 2; P is R^(c)—C(O)—; R^(c) is R^(D); R^(D) is substituted or unsubstituted mono- or bicyclic ring system selected from the group consisting of phenyl, pyridinyl, pyrimidinyl, naphthyl, benzimidazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, tetrahydroquinoxalinyl, and dihydrobenzoxazinyl; R^(a) is hydrogen, C₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic, —(CH₂)_(m)—CH₂—R^(B), —(CH₂)_(m)—CH₂—NHC(═NR⁴)NH—Y, —(CH₂)_(m)—CH₂—CON(R⁴)₂, —(CH₂)_(m)—CH₂—N(R⁴)CON(R⁴)₂, —(CH₂)_(m)—CH(R⁶)N(R⁴)₂, —(CH₂)_(m)—CH(R^(5a))—OR^(5b), or —(CH₂)_(m)—CH(R⁵)—SR⁵; R^(a1) is hydrogen, C₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic, —(CH₂)_(m)—CH₂—R^(B), —(CH₂)_(m)—CH₂—NHC(═NR⁴)NH—Y, —(CH₂)_(m)—CH₂—CON(R⁴)₂, —(CH₂)_(m)—CH₂—N(R⁴)CON(R⁴)₂, —(CH₂)_(m)—CH(R⁶)N(R⁴)₂, —(CH₂)_(m)—CH(R^(5a))—OR^(5b), or —(CH₂)_(m)—CH(R⁵)—SR⁵; each R^(a2) independently is hydrogen, C₁₋₆ aliphatic, C₁₋₄ fluoroaliphatic, —(CH₂)_(m)—CH₂—R^(B), —(CH₂)_(m)—CH₂—NHC(═NR⁴)NH—Y, —(CH₂)_(m)—CH₂—CON(R⁴)₂, —(CH₂)_(m)—CH₂—N(R⁴)CON(R⁴)₂, —(CH₂)_(m)—CH(R⁶)N(R⁴)₂, —(CH₂)_(m)—CH(R^(5a))—OR^(5b), or —(CH₂)_(m)—CH(R⁵)—SR⁵; each R^(B) independently is a substituted or unsubstituted mono- or bicyclic ring system; each R⁴ independently is hydrogen or a substituted or unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; or two R⁴ on the same nitrogen atom, taken together with the nitrogen atom, form a substituted or unsubstituted 4- to 8-membered heterocyclyl ring having, in addition to the nitrogen atom, 0-2 ring heteroatoms independently selected from N, O, and S; each R⁵ independently is hydrogen or a substituted or unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; each R^(5a) independently is hydrogen or a substituted or unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; each R^(5b) independently is hydrogen or a substituted or unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; each R⁶ independently is a substituted or unsubstituted aliphatic, aryl, or heteroaryl group; Y is hydrogen, —CN, or —NO₂; m is 0, 1, or 2; each of R^(b1) and R^(b2) independently is hydrogen, —CO₂H, —OH, or a substituted or unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; each of R^(b3) and R^(b4) independently is hydrogen, —CO₂H, or a substituted or unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; or R^(b2) and R^(b4) are each independently hydrogen, and R^(b1) and R^(b3), taken together with the carbon atoms to which they are attached, form an unsubstituted or substituted fused 4- to 8-membered non-aromatic ring having 0-3 ring heteroatoms selected from the group consisting of O, N, and S, wherein said ring may be optionally fused to an unsubstituted or substituted 4- to 8-membered non-aromatic ring, or 5- to 6-membered aromatic ring having 0-3 ring heteroatoms selected from the group consisting of O, N, and S; or R^(b2) and R^(b4) are absent, and R^(b1) and R^(b3), taken together with the carbon atoms to which they are attached, form an unsubstituted or substituted fused 5- to 6-membered aromatic ring having 0-3 ring heteroatoms selected from the group consisting of O, N, and S, wherein said ring may be optionally fused to an unsubstituted or substituted 4- to 8-membered non-aromatic ring, or 5- to 6-membered aromatic ring having 0-3 ring heteroatoms selected from the group consisting of O, N, and S; and n is 0 or 1; the process comprising: (1) coupling a compound of Formula (i) with a compound of Formula (ii) to form a compound of Formula (iii)

wherein X⁻ is a counter anion; and PG is hydrogen or a protecting group; (2) deprotecting the compound of Formula (iii) to form a compound of Formula (iiia)

(3) coupling the compound of Formula (iiia) with a compound, P-LG, to form a compound of Formula (iv)

wherein LG is a leaving group; (4) deprotecting the compound of Formula (iv) to form the compound of Formula (v)

(5) reacting the compound of Formula (v) with an alpha hydroxy carboxylic acid or a beta hydroxy carboxylic acid to form a compound of Formula (II).
 2. The process of claim 1, wherein the reaction of step (1) or step (3) or both is conducted in the presence of a peptide coupling reagent.
 3. The process of claim 2, wherein the peptide coupling reagent is selected from the group consisting of a carbodiimide reagent, phosphonium reagent, and uronium reagent.
 4. The process of claim 3, wherein the peptide coupling reagent is selected from one or more of the group consisting of dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), and O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU).
 5. The process of claim 1, further comprising converting the carboxylic acid moiety of compound (i) to an activated ester or acid halide prior to the reaction of step (1).
 6. The process of claim 5, wherein said activated ester or acid halide is an O—(N-hydroxysuccinimide) ester.
 7. The process of claim 1, wherein the reaction of step (3) is conducted in the presence of a solvent.
 8. The process of claim 7, wherein the solvent is tetrahydrofuran.
 9. The process of claim 1, wherein the reaction of step (4) is conducted in the presence of an aqueous mineral acid.
 10. The process of claim 9, wherein the mineral acid is hydrochloric acid.
 11. The process of claim 1, wherein the reaction of step (4) is conducted in the presence of an organic boronic acid acceptor.
 12. The process of claim 11, wherein the organic boronic acid acceptor is i-BuB(OH)₂.
 13. The process of claim 1, wherein the reaction of step (5) is conducted in the presence of a solvent selected from the group consisting of ethyl acetate, methyl isobutyl ketone, acetone, acetonitrile, 2-methyltetrahydrofuran, anisole, isopropyl acetate, dimethoxyethane, tetrahydrofuran, dioxane, dichloromethane, toluene, heptane, methyl-cyclohexane, tert-butylmethyl ether, and any combination thereof.
 14. The process of claim 1, wherein the reaction of step (5) is conducted in the presence of a catalyst, wherein the catalyst is an organic amine base.
 15. The process of claim 14, wherein the catalyst is selected from the group consisting of triethylamine, triethylenediamine, pyridine, collidine, 2,6-lutidine, 4-dimethylaminopyridine, di-tertbutylpyridine, N-methylmorpholine, N-methylpiperidine, tetramethylguanidine, diazabicyclo[5.4.0]undec-7-ene (DBU), 1,4-diazabicyclo[2.2.2]octane, 1,5-diazabicycle[4.3.0]non-5-ene, N,N′diisopropylethylamine, or any combination thereof.
 16. The process of claim 1, wherein step (5) further comprises cooling the solution.
 17. The process of claim 16, wherein the cooling comprises cooling the solution uncontrolled until the internal temperature is about 25° C.
 18. The process of claim 1, wherein the reaction of step (5) is conducted in the presence of a co-solvent.
 19. The process of claim 18, wherein the co-solvent is selected from the group consisting of heptane, methylcyclohexane, toluene, tert-butylmethyl ether, ethyl acetate, or any combination thereof.
 20. The process of claim 1, further comprising (6) isolating the compound of Formula (II) as a crystalline solid.
 21. The process of claim 1, wherein PG is a protecting group selected from the group consisting of an acyl protecting group and a urethane protecting group.
 22. The process of claim 21, wherein PG is a protecting group selected from the group consisting of formyl, acetyl, succinyl, methoxysuccinyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and fluorenylmethoxycarbonyl (Fmoc).
 23. The process of claim 1, wherein LG is —Cl.
 24. The process of claim 1, wherein X⁻ is CF₃CO₂ ⁻.
 25. The process of claim 1, wherein R^(D) is 2,5-dichlorophenyl.
 26. The process of claim 1, wherein A is 0; R^(a) is C₁₋₆ aliphatic; R^(a1) is hydrogen, C₁₋₆ aliphatic, —(CH₂)_(m)—CH₂—R^(B), or —(CH₂)_(m)—CH(R^(5a))—OR^(5b); each of R^(b1) and R^(b2) independently is hydrogen, —CO₂H, —OH, or a substituted or unsubstituted aliphatic; each of R^(b3) and R^(b4) independently is hydrogen, C₁₋₆ aliphatic, or —(CH₂)_(p)CO₂H; and p is 0 or
 1. 27. The process of claim 1, wherein A is 0; R^(D) is 2,5-dichlorophenyl; R^(a) is isobutyl; R^(a1) is hydrogen; each R^(b1) and R^(b2) independently is hydrogen; each R^(b3) and R^(b4) independently is —(CH₂)_(p)—CO₂H; p is 0 or 1; and n is 0 or
 1. 28. The process of claim 1, wherein the alpha hydroxy carboxylic acid or a beta hydroxy carboxylic acid is citric acid.
 29. The process of claim 1, wherein the compound of Formula (II) is 4-(R,S)-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylic acid.
 30. The process of claim 1, wherein the compound of Formula (II) is 2,2′-{2-[(1R)-1-({[(2,5-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]-5-oxo-1,3,2-dioxaborolane-4,4-diyl}diacetic acid.
 31. A process for generating a compound of Formula (II)

or a pharmaceutically acceptable salt thereof, wherein A is 0, 1, or 2; P is R^(c)—C(O)—; R^(c) is R^(D); R^(D) is substituted or unsubstituted mono- or bicyclic ring system selected from the group consisting of phenyl, pyridinyl, pyrimidinyl, naphthyl, benzimidazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, tetrahydroquinoxalinyl, and dihydrobenzoxazinyl; R^(a) is hydrogen, C₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic, —(CH₂)_(m)—CH₂—R^(B), —(CH₂)_(m)—CH₂—NHC(═NR⁴)NH—Y, —(CH₂)_(m)—CH₂—CON(R⁴)₂, —(CH₂)_(m)—CH₂—N(R⁴)CON(R⁴)₂, —(CH₂)_(m)—CH(R⁶)N(R⁴)₂, —(CH₂)_(m)—CH(R^(5a))—OR^(5b), or —(CH₂)_(m)—CH(R⁵)—SR⁵; R^(a1) is hydrogen, C₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic, —(CH₂)_(m)—CH₂—R^(B), —(CH₂)_(m)—CH₂—NHC(═NR⁴)NH—Y, —(CH₂)_(m)—CH₂—CON(R⁴)₂, —(CH₂)_(m)—CH₂—N(R⁴)CON(R⁴)₂, —(CH₂)_(m)—CH(R⁶)N(R⁴)₂, —(CH₂)_(m)—CH(R^(5a))—OR^(5b), or —(CH₂)_(m)—CH(R⁵)—SR⁵; each R^(a2) independently is hydrogen, C₁₋₆ aliphatic, C₁₋₆ fluoroaliphatic, —(CH₂)_(m)—CH₂—R^(B), —(CH₂)_(m)—CH₂—NHC(═NR⁴)NH—Y, —(CH₂)_(m)—CH₂—CON(R⁴)₂, —(CH₂)_(m)—CH₂—N(R⁴)CON(R⁴)₂, —(CH₂)_(m)—CH(R⁶)N(R⁴)₂, —(CH₂)_(m)—CH(R^(5a))—OR^(5b), or —(CH₂)_(m)—CH(R⁵)—SR⁵; each R^(B) independently is a substituted or unsubstituted mono- or bicyclic ring system; each R⁴ independently is hydrogen or a substituted or unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; or two R⁴ on the same nitrogen atom, taken together with the nitrogen atom, form a substituted or unsubstituted 4- to 8-membered heterocyclyl ring having, in addition to the nitrogen atom, 0-2 ring heteroatoms independently selected from N, O, and S; each R⁵ independently is hydrogen or a substituted or unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; each R^(5a) independently is hydrogen or a substituted or unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; each R^(5b) independently is hydrogen or a substituted or unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; each R⁶ independently is a substituted or unsubstituted aliphatic, aryl, or heteroaryl group; Y is hydrogen, —CN, or —NO₂; m is 0, 1, or 2; each of R^(b1) and R^(b2) independently is hydrogen, —CO₂H, —OH, or a substituted or unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; each of R^(b3) and R^(b4) independently is hydrogen, —CO₂H, or a substituted or unsubstituted aliphatic, aryl, heteroaryl, or heterocyclyl group; or R^(b2) and R^(b4) are each independently hydrogen, and R^(b1) and R^(b3), taken together with the carbon atoms to which they are attached, form an unsubstituted or substituted fused 4- to 8-membered non-aromatic ring having 0-3 ring heteroatoms selected from the group consisting of O, N, and S, wherein said ring may be optionally fused to an unsubstituted or substituted 4- to 8-membered non-aromatic ring, or 5- to 6-membered aromatic ring having 0-3 ring heteroatoms selected from the group consisting of O, N, and S; or R^(b2) and R^(b4) are absent, and R^(b1) and R^(b3), taken together with the carbon atoms to which they are attached, form an unsubstituted or substituted fused 5- to 6-membered aromatic ring having 0-3 ring heteroatoms selected from the group consisting of O, N, and S, wherein said ring may be optionally fused to an unsubstituted or substituted 4- to 8-membered non-aromatic ring, or 5- to 6-membered aromatic ring having 0-3 ring heteroatoms selected from the group consisting of O, N, and S; and n is 0 or 1; the process comprising: (1a) coupling a compound of Formula (vi) with a compound, P-LG, to form a compound of Formula (vii)

wherein PG is hydrogen or a protecting group; and LG is a leaving group; (2a) deprotecting the compound of Formula (vii) to form a compound of Formula (viia)

(3a) coupling the compound of Formula (viia) with a compound of Formula (i) to form a compound of Formula (iv)

wherein X⁻ is a counter anion; (4) deprotecting the compound of Formula (iv) to form a compound of Formula (v)

 and (5) reacting the compound of Formula (v) with an alpha hydroxy carboxylic acid or a beta hydroxy carboxylic acid to form a compound of Formula (II). 